Construction of stringent gene content matrices was accomplished for 21 Anopheline mosquito species and strains and four outgroups species. The presence absence matrix using e−75 as a cutoff in single linkage clustering had over 17,000 ortholog groups. We used the gene content matrix to generate a phylogenetic hypothesis that is in general agreement with gene sequence based phylogenies. In addition to establishing a congruent gene content phylogeny we examined the consistency of three methods for analyzing presence absence data – unweighted parsimony, dollo parsimonly and maximum likelihood using a BINGAMMA model. An examination of the chromosomal location of the gains and losses in the presence absence matrix revealed a low frequency of gains and losses at centromeres and tips of chromosomes.