Simple Summary All Poropuntiinae fish species are diploid and have 50 chromosomes in their cells; however, their karyotypes differ (the organization of chromosomes according to size and shape). The goal of this study is to compare the genomic differences between their conserved karyotypes using conventional and molecular cytogenetic methods. We found distinct patterns in the distribution of ribosomal DNA and microsatellites, indicating that, while their karyotypes are conserved, these fishes have species-specific patterns. Our comparative genomic hybridization experiment reveals that any of their repetitive DNA content matches, highlighting the differences between such species. This study adds to our understanding of chromosome evolution in Cyprinidae fishes, which include diploid, tetraploid, and hexaploid species. Abstract The representatives of cyprinid lineage ‘Poropuntiinae’ with 16 recognized genera and around 100 species form a significant part of Southeast Asian ichthyofauna. Cytogenetics are valuable when studying fish evolution, especially the dynamics of repetitive DNAs, such as ribosomal DNAs (5S and 18S) and microsatellites, that can vary between species. Here, karyotypes of seven ‘poropuntiin’ species, namely Cosmochilus harmandi , Cyclocheilichthys apogon , Hypsibarbus malcomi , H. wetmorei , Mystacoleucus chilopterus , M. ectypus , and Puntioplties proctozysron occurring in Thailand were examined using conventional and molecular cytogenetic protocols. Variable numbers of uni- and bi-armed chromosomes indicated widespread chromosome rearrangements with a stable diploid chromosome number (2n) of 50. Examination with fluorescence in situ hybridization using major and minor ribosomal probes showed that Cosmochilus harmandi , Cyclocheilichthys apogon , and Puntioplites proctozystron all had one chromosomal pair with 5S rDNA sites. However, more than two sites were found in Hypsibarbus malcolmi , H. wetmorei , Mystacoleucus chilopterus , and M. ectypus . The number of chromosomes with 18S rDNA sites varied amongst their karyotypes from one to three; additionally, comparative genomic hybridization and microsatellite patterns varied among species. Our results reinforce the trend of chromosomal evolution in cyprinifom fishes, with major chromosomal rearrangements, while conserving their 2n.