The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior-posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.