Squamata is the most diversified reptilian order that has been traditionally classified into three suborders – Lacertilia, Serpentes and Amphisbaenia in which Lacertilia have about 16-19 families. But the phylogenetic relationships among major groups of Lacertilia remain controversial. In this paper, the complete mitochondrial DNA sequence of Eremias brenchleyi was determined by using long-and-accurate PCR (LA-PCR). The mtDNA sequence is 19 542 bp, making it the longest mitochondrial genome in squamates species reported so far. It shows the typical vertebrate arrangement of genes. The control region of E. brenchleyi was characterized by two conspicuous 65 bp and 56 bp tandem repeats at its 5′ and 3′ terminus respectively. In order to study the higher level relationships of squamates, the phylogenetic study including all currently available squamates mitochondrial sequences was carried out. We obtained a relationship of 16 families of lizards (Lacertidae, Scincidae, Iguanidae, Chameleonidae, Agamidae, Trogonophidae, Bipedidae, Shinisauridae, Helodermatidae, Amphisbaenidae, Gekkonidae, Varanidae, Anguidae, Xantusiidae, Rhineuridae, Cordylidae) and 8 families of Serpentes. The internal relationships within this group yielded high bootstrap support and were more congruent with morphological analyses.