The suborder Serpentes is divided into 2 infraorders, Scolecophidia and Alethinophidia, which diverged at an early stage of snake diversification. In this study, we examined karyotypes of 4 scolecophidian species (Letheobia simonii, Xerotyphlops vermicularis, Indotyphlops braminus, and Myriopholis macrorhyncha) and performed FISH with 18S-28S rDNA as well as microchromosomal and Z chromosome-linked genes of Elaphe quadrivirgata (Alethinophidia) to investigate the karyotype evolution in the scolecophidian lineage. Diploid chromosome numbers of X. vermicularis and L. simonii were 30 (16 macrochromosomes and 14 microchromosomes) and 32 (16 macrochromosomes and 16 microchromosomes), respectively. The karyotype of a female M. macrorhyncha consisted of 15 macrochromosomes and 19 microchromosomes, including a heterochromatic microchromosome, indicating the presence of a heteromorphic chromosome pair. E. quadrivirgata Z-linked genes mapped to chromosome 4 of M. macrorhyncha, not to the heteromorphic pair. Therefore, M. macrorhyncha may have differentiated ZW sex chromosomes which are not homologous to those of E. quadrivirgata. One of the E. quadrivirgata microchromosomal genes mapped to the terminal region of chromosome 4q in X. vermicularis, suggesting that fusions between microchromosomes and macrochromosomes occurred in this species. rDNA was localized in different macrochromosomal pairs in the 2 diploid scolecophidian snakes examined here, whereas the gene location in a microchromosomal pair was conserved in 5 alethinophidian species examined. These results might imply the occurrence of chromosome fusions in the scolecophidian lineages. In I. braminus, a unique parthenogenetic snake with a triploid karyotype (21 macrochromosomes and 21 microchromosomes), morphological heteromorphisms were identified in chromosomes 1 and 7. Such heteromorphisms in 2 chromosomes were also observed in individuals from distant locations in the broad distribution range of this species, suggesting that the heteromorphisms were fixed in the genome at an early stage of its speciation.