The mitochondrial DNA (mtDNA) control region (CR) is a potentially informative target for studying the structure and evolution of non-coding sequences and the conservation of functional homologies in the insect mitochondrial genome. Specific features of CR include a highly biased AT content, putative regulatory elements for the transcription/replication process, high mutation levels and common rearrangements of specific sequence elements and flanking genes. The aim of this work is the molecular characterization of the entire mtDNA CR and its flanking genes for five Muscidae flies, including species of great medical and veterinary importance, Haematobia irritans (horn fly), Musca domestica (house fly), Stomoxys calcitrans (stable fly), Ophyra aenescens and Atherigona orientalis. Conserved elements of the insect mtDNA CR (such as poly-T streches, TA(A)n in tandem repeats, ‘stem-and-loop’ structures and G+C-rich regions) and conserved-sequence-blocks previously described for myiasis-causing flies have also been identified in the Muscidae CR. The conservation of CR structural homologies among these species suggests that Muscoidea and Oestroidea may share a similar regulatory system for mtDNA transcription/replication processes. The analysis of S.calcitrans CR was optimized by the design of a new set of specific primers and preliminary results indicate different patterns of structural organization regarding its flanking genes. Preliminary analysis of CR sequences polymorphisms of H.irritans samples from Brazil, Argentina, Uruguay, Venezuela and Malaysia shows high levels of nucleotide substitutions comparing South America vs Asian samples. Significant length variation of CR sequences was also identified in these population samples. Financial support: PROFIX/CNPq, FAPESP.