Genetic variability was assessed among three species of genus Channa, namely C. gachua, C. punctata and C. striata, using karyological tools including karyomorphology, silver nitrate and chromomycin A 3 staining of nucleolar organizer regions (NORs) and C-banding. Additionally, sequence variation in partial 18S and divergent domain 1 of 28S ribosomal DNA was utilized for inter-species diversity assessment. Diploid chromosome numbers of 32, 40 and 52 in the three species were observed and the karyotype formula was derived as 18m + 12sm + 2st (fundamental arm number [FN] = 62); 6m + 2sm + 10st + 22t (FN = 48) and 12m + 10sm + 14st + 16t (FN = 86) in C. punctata, C. striata and C. gachua, respectively. Similarly, variations were also observed in number and location of silver and chromomycin A 3 stained NORs and C-banding patterns. DNA sequencing generated sizes for 18S of 243, 248, 245 bp and for 28S of 401, 392 and 393 bp in C. gachua, C. punctata and C. striata, respectively. The results indicated that the cytogenetic and molecular markers are useful for the assessment of genetic diversity among the fish species and could provide valuable information for germplasm evaluation and ex situ conservation.