The African malaria mosquito Anopheles gambiae is the major vector of Plasmodium falciparum parasite in Africa transmitting malarial disease from person to person by its blood feeding almost exclusively from humans. The southern house mosquito,Culex quiqefasiatus, is an important mosquito vector of viruses such as West Nile virus,St. Louis encephalitis virus, and nematodes that cause lymphatic filarisis. Because these two mosquitoes greatly impact public health issues, their genomes have been sequenced; however, comprehensive genome sequence assemblies are not available due to these factors: 1) whole genome physical maps have not been constructed for informed scaffold construction and validations, and 2) abundance of repetitive sequences and pervasive genomic heterozygosity complicate genome assembly efforts. We report here our efforts to construct optical maps for these mosquito genomes and their utility for the construction of comprehensive and validated genome sequence assemblies. Our laboratory has developed the Optical Mapping System, which is the first single molecule platform to provide a detailed and comprehensive analysis of any complex genome. These features complement sequencing efforts especially for the next generation sequencing data through the construction of physical scaffolds that order and place nascent contigs within a whole-genome context. As such, we present here two de novo physical maps for the Anopheles gambiae and the Culexquiqefasiatus, and demonstrate how optical map resources have been integrated into these sequencing projects. Our optical map of Anopheles gambiae genome comprises 26 contigs, averaging 11.7 Mb, and comparison with the current build of Anopheles gambiae sequence scaffolds shows 56% congruency between sequence scaffolds and optical maps, pinpoints sequence misassemblies, and characterizes the sequence gaps. Our optical map of Culex quiquefasiatus genome consists of 91 optical map contigs, averaging 6.5 Mb, and comparison with the current sequence scaffolds shows only 9.4% congruency between sequence scaffolds and optical maps presumably due to the fragmentations of genome sequence assembly caused by the abundance of repetitive transposable elements in Culex genome. Therefore, optical maps not only accelerate sequence assembly efforts for accurate reference genome sequence build for comparative genomic studies, but also importantly provide an unique resource for the fine-scale dissection of complex genome structures that are frequently missed using next-generation sequencing approaches.