Of the total 47 species in the subgenus Acanthosentis, 43 have been reported from the freshwater fishes of Asia. Amin et al. (2017) provided a key to the 23 species of the genus Acanthogyrus reported from the Indian subcontinent. The present study reports two new species: Acanthogyrus bispinosa n. sp. and A. garciai n. sp. from Cirrhinus mrigala Hamilton and Labeo calbasu Hamilton, respectively, and two previously described species: A. golvani Gupta and Jain, 1980 and A. hereterospinus Khan and Bilqees, 1990 from L. rohita Hamilton and L. catla Hamilton, respectively. A. bispinosa n. sp. comprises 3 circles of 6 proboscis hooks each. Trunk spines in A. bispinosa n. sp are divided into two groups: anterior and posterior separated by unarmed region, which has not been previously reported in the subgenus. Anterior spines are present in 7–8 and 7–10 circles in females and males, respectively, whereas posterior spines are in 23–28 and 31–38 circles in males and females, respectively. A. garciai n. sp. comprises 3 circles of 6–8 hooks each and a single set of trunk spines is present in A. garciai n. sp., comprising 35–42 and 25–45 circles in males and females, respectively. All four species were also characterised based on the 18S, 28S, and ITS1-5.8S-ITS2 rRNA molecular markers. The Bayesian inference tree generated based on these markers showed distinct identities of all the species, with a significant molecular divergence, ranging from 3.2 to 53.6%.