SummaryThe first cytogenetics of the puff-faced water snake (Homalopsis buccata) from Khon Kaen Province, Thailand was obtained from the present study. Mitotic chromosome was prepared directly from the spleens of specimens after in vivo colchicines treatment. The metaphase spreads were performed on microscopic slides and air-dried. Conventional staining, Ag-NOR banding, and GTG-banding techniques were applied to stain the chromosome with Giemsa s solution. Results showed that the number of diploid chromosome was 2n=36, while the fundamental number (NF) was 52 in both males and females. The types of macrochromosome were four large metacentric, two medium metacentric, six small metacentric, and two small acrocentric chromosome, and 20 microchromosomes. It was found that nucleolar organizer regions (NORs, the representative of chromosome marker) locate on the long arms near centromeres of a pair of microchromosomes, pair 15. The Z chromosome was a small metacentric chromosome, while the W chromosome was a small submetacentric chromosome. From the GTG-banding technique, it was found that each chromosome pairs could be clearly differentiated and the numbers of bands in the H. buccata was 109. The karyotype formula is as follows:2n (