The ISSR-PCR and karyological analysis methods are successfully used to detect the genetic variations between the different species of fishes, especially these belonging to the same family. The aim of this study was to determine the karyotypes and molecular genetic variations of three species of family Osphronemidae, Trichogaster trichopterus, Trichogaster leeri and Colisa laliaby using ISSR-PCR and chromosomal analysis. Samples were collected from ornamental fish farms in Egypt. The diploid chromosome numbers and Fundamental numbers for species under study were 2n = 46 and FN = 46, 2n = 46 and FN = 46, 2n = 46 and FN = 70 respectively, the first two species have same karyotype but the third species was different. The ISSR-PCR analysis was carried out using ten primers. All primers were successfully amplified on the genomic DNA extracted from all studied fish species. These findings indicate that ISSR-PCR and cytogenetic analysis are very useful in determination of genetic molecular variations and relationship degree between the species which belong to same family.