Darevskia rostombekowi , the most outstanding of the seven known parthenogenetic species in the genus Darevskia , is the result of an ancestral cross between two bisexual species Darevskia raddei and Darevskia portschinskii . The chromosomal set of this species includes a unique submetacentric autosomal chromosome; the origin of this chromosome was unresolved as only acrocentric chromosomes are described in the karyotypes of Darevskia genus normally. Here, we applied a suite of molecular cytogenetic techniques, including the mapping of telomeric (TTAGGG) n repeats using fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and whole-chromosome painting (WCP) in both D. rostombekowi and parental ( D. portschinskii and D. raddei ) species. The obtained results in total suggest that a de novo chromosomal rearrangement via Robertsonian translocation (centric fusion) between two maternal ( D. raddei ) acrocentric chromosomes of different size was involved in the formation of this unique submetacentric chromosome present in the parthenogenetic species D. rostombekowi . Our findings provide new data in specific and rapid evolutional processes of a unisexual reptile species karyotype.