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Spalerosophis Jan, 1865, is a colubrid snake genus distributed in arid and semiarid regions from northwestern Africa to northern India. Six species of Spalerosophis are identified based on traditional morphology; however, there are arguments about their systematic status. We performed the first molecular phylogenetic analyses for this genus using nucleotide sequences of c. 2278 bp from three mitochondrial genes (12S rRNA, 16S rRNA, and cyt b) to investigate the phylogenetic relationships and historical evolution of the poorly known Spalerosophis species and doubtful populations. We used fossil calibrations for dating divergences. Our phylogenetic analyses clearly showed five separate species with high support values, including S. arenarius, S. atriceps, S. microlepis, S. diadema schirasianus, and S. diadema cliffordii. Bayesian calibrated molecular clock suggested that the genus Spalerosophis diverged from the common ancestor in the Paleogene around 35.28 Mya (25.89–44.66). Our analysis indicated that isolated S. atriceps was separated around 21.72 Mya (10.86–33.04) as the basal diverged species of the genus. This suggested that the species of Spalerosophis probably originated from an ancestor somewhere in the Iranian Plateau and then dispersed to its current geographical range. Spalerosophis arenarius and S. atriceps were identified as the two last diverged members of the genus, separated from S. d. schirasianus during the Middle Miocene about 13.31 Mya (5.52–22.33). These results indicate that the separation of Spalerosophis species coincided with the orogenic events of the Zagros Mountains in western Iran and the Sulaiman Mountains in Afghanistan, India, and Pakistan. Their diversification may therefore be the result of vicariance events that promoted their current distribution. This work provides a foundation for future studies on the phylogeny, diversity, and evolution of the genus Spalerosophis and highlights the need for more molecular studies on unknown snakes. [ABSTRACT FROM AUTHOR]