Entries with (?) after the number indicate brotherness of dubious canonicity

SUPER SMASH BROS. (N64):

1. Mario (Brother of Luigi)

2. (?) Donkey Kong (Diddy Kong is referred to as Donkey Kong's nephew in some material, implying that DK has a sibling. However other more recent works refer to Diddy as just his friend. so this may have been retconned)

3. Luigi (Brother of Mario)

4. Ness (Has a younger sister)

TOTAL BROTHER COUNT: Definite 3/12 (25% of playable characters) - Potential 4/12 (33% of playable characters)

SUPER SMASH BROS. MELEE (GCN):

5. (?) Bowser (In world 8-4 of the Japanese Super Mario Bros. 2 (released as Super Mario Bros.: The Lost Levels outside of Japan), in addition to fighting Bowser there is also a 2nd Bowser with a blue coloration earlier in the level, which some secondary materials refer to as "Bowser's Brother". However other materials refer to him as just a "Fake Bowser", so this may not be canon. One of Bowser's selectable colour schemes in Smash 4 and Ultimate is a blue colour scheme that may be a reference to Bowser's Brother, so this may count as an additional brother for those games if you choose to count it.)

6. (?) Dr. Mario. (Potential brother of Dr. Luigi. There's some question over whether Dr. Mario is the same character as Mario or not, and if they are separate characters that makes it uncertain as to whether Dr. Luigi is related to Dr. Mario in the same way as regular Luigi is to regular Mario. The official english Nintendo store page for the game Dr. Luigi says the titular character is "taking over responsibilities from his brother Mario", which seems to imply they are the same characters and thus still brothers.)

7. Marth (Has an older sister)

TOTAL BROTHER COUNT: Definite 4/25 (16% of playable characters) - Potential 7/25 (28% of playable characters)

SUPER SMASH BROS. BRAWL (WII):

8. (?) Wario (Potential brother of Waluigi. Some secondary material has referred to them as brothers, however other sources and comments from developers have claimed they are not related)

9. (?) Snake (Brother of Liquid and Solidus Snake. The name being just "Snake" leaves some ambiguity as to whether he is Solid Snake or a young Naked Snake / Big Boss, however his default costume and trophy in Brawl seem to imply this incarnation is specifically meant to be Solid Snake. Additionally, it could be argued that since they were the result of cloning they may not count as truly brothers. Liquid refers to Solid as his brother multiple times, but I could not find any instances of Solid referring to Liquid as his brother, so it's unclear if he personally considers themselves to be brothers or not.)

10. Ike (Has a younger sister)

11. Lucas (Has an older twin brother)

12. Toon Link (Based on the Wind Waker incarnation of Link, who has a younger sister)

TOTAL BROTHER COUNT: Definite 7/35 (20% of playable characters) - Potential 12/35 (34%)

SUPER SMASH BROS. 4 (WII U & 3DS):

(Snake did not return for this game, removing 1 potential brother from the count)

13. Mega Man (Has a younger sister, though it could be argued since they're both robots they may not count as true siblings)

14 - 19. Koopalings (Except Wendy, who is a sister. While the Koopalings were originally referred to as Bowser's children, this has been officially retconned by the time of the introduction of Bowser Jr., meaning that he does not count as a brother.)

TOTAL BROTHER COUNT: Definite 14/65 (21% of playable characters) - Potential 18/65 (27% of playable characters)

SUPER SMASH BROS. ULTIMATE (SWITCH) / Series Total:

20. Chrom (Has both an older and younger sister)

21. Corrin (Has several half, step, and adoptive brothers and sisters, making the male skin of this character a brother)

22. Richter Belmont (Has an adopted younger sister)

23. (?) King K. Rool (an official character page on Nintendo's Japanese website for Donkey Kong 64 refers to K. Lumsy from that game as K. Rool's younger brother. This is neither confirmed nor contradicted within the game itself and K. Lumsy is never referenced in the series after this.)

24. Banjo (Has a younger sister.)

25. Terry Bogard (Has a younger brother.)

26. Kazuya Mishima (Has an adopted brother, a half-brother and a half-sister)

TOTAL BROTHER COUNT: Definite 20/89 (22% of playable characters) - Potential 26/89 (29% of playable characters)