Sea turtles are considered flagship species for marine biodiversity conservation and are considered to be at varying risk of extinction globally. Cases of hybridism have been reported in sea turtles, but chromosomal analyses are limited to classical karyotype descriptions and a few molecular cytogenetic studies. In order to compare karyotypes and understand evolutive mechanisms related to chromosome dif-ferentiation in this group, Chelonia mydas, Caretta caretta, Eretmochelys imbricata, and Lepidochelys olivacea were cytogenetically characterized in the present study. When the obtained cytogenetic data were compared with the putative ancestral Cryptodira karyotype, the studied species showed the same diploid number (2n) of 56 chromosomes, with some variations in chromosomal morphology (karyotypic formula) and minor changes in longitudinal band locations. In situ localization using a 18S ribosomal DNA probe indicated a homeologous microchromosome pair bearing a 45S ribosomal DNA locus and size heteromorphism in all 4 species. Interstitial telomeric sites were identified in a microchromosome pair in C. mydas and C. caretta. The data showed that interspecific variations occurred in chromosomal sets among the Cheloniidae species, in addition to other Cryptodira karyotypes. These variations generated lineage-specific karyotypic diversification in sea turtles, which will have considerable implications for hybrid recognition and for the study, the biology, ecology, and evolutionary history of regional and global populations. Furthermore, we demonstrated that some chromosome rearrangements occurred in sea turtle species, which is in conflict with the hypothesis of conserved karyotypes in this group.