C-mos is a highly conserved intronless gene that has proved useful in the analysis of ancient phylogenetic relationships within vertebrates. We selected the Iberian endemic Schreiber's green lizard (Lacerta schreiberi) that persisted in allopatric refugia since the late Pliocene to investigate the utility of the C-mos nuclear gene for intraspecific phylogeographic studies. Our combination of DNA sequencing with the high resolving power of single-strand conformational polymorphism (SSCP) effectively discriminated four common alleles showing strong population structuring (FST = 0.46). In addition, reconstruction of allele phylogenetic relationships further improved our understanding of C-mos spatial patterns of variation and allowed a comparison with previously described mitochondrial DNA data. Finally, limited sequencing of an extended C-mos fragment in six additional Lacerta species showed extensive polymorphism, to our knowledge representing a rare example of variation in a highly conserved nuclear gene.