We have cloned and sequenced an orthologue of the human SOX9 gene in the lizard, Calotes versicolor, which lacks heteromorphic sex chromosomes as well as temperature-dependent sex determination. PCR amplification of Calotes cDNA using human SOX9 primers yielded an 861-bp fragment which harboured the high-mobility group domain and flanking sequences and had identity greater than 85% at nucleotide and greater than 90% at amino acid level with the hitherto known homologues of Sox9, demonstrating its evolutionary conservation. CvSox9 makes an approx. 5-kb transcript which is expressed in the genital ridge and mullerian duct of all the embryos from their very inception. In the genital ridge the expression in day 40 and day 45 embryos is confined to the embryonic testis (medulla) of only 50% embryos, indicating a shift in the expression from non-gonad-specific to testis-specific. The temporal and cell-specific expression in the genital ridge suggests a vital role for CvSox9 in the determination and/or differentiation of testis.