The mitochondrial DNA loci Cytochrome Oxidase I, (COI), Intergenic Spacer (IGS), tRNA for Leucine (tRNALeu) and Cytochrome Oxidase II (COII) have been largely used in phylogenetic and population analysis. In order to proceed population studies in the ant Atta laevigata (Attini: Formicidae), we sequenced 534 base pairs of the COI-IGS-tRNALeu-COII loci in 40 specimens of this ant living in the Northwest region of São Paulo state. Phylogenetic analysis of 120 parsimony-informative characters of the generated sequences supports the existence of two distinct A. laevigata populations, which were named A and B. We observed the following differences in gene sequences between populations A and B: Synonymous transitions in third codon position in COI (2 substitutions) or in COII (4 substitutions); One synonymous transversion in a third codon position in COI; One non-synonymous transition in a first codon position in COII, which resulted in the coding of an Alanine in population A or a Threonine in population B; 32 1-nucleotide indels in tRNALeu; 1- to 30- contiguous nucleotide indels in IGS. Our results show that COI, IGS, tRNALeu and COII are useful markers for population studies in Atta laevigata, and that two populations of this ant can be found in São Paulo state.