Diseases like malaria claim the lives of millions of people every year. This deadly disease can result in considerable morbidity, which presently affects countries in Africa, Eurasia, and South America. Anopheline mosquitoes transmit this disease. Studies look at the identification first of the species to accurately determine their distribution. For identification, sequencing of the Internal Transcribed Spacer 2 region of the ribosomal DNA is analyzed. Although African Anophelines are very well studied species, there has been no recent significant research for Holarctic Anophelines. In particular, in North America and Eurasia, because of the eradication of malaria in the northern territories and the focus on other diseases transmitted by Aedes and Culex mosquitoes. In this study, we first look at the Holarctic species Anopheles punctipennis in North America from the Midwest to the eastern United States. Then we look at samples received from Eurasia, in particular Russia and Iran. We physically harvested 500 mosquitoes from ten breeding sites to analyze the identity and distribution of An. punctipennis. We received 110 samples from Russia and 180 samples from Iran to examine the identification and geographic distribution of An. daciae and An. persiensis. Mosquito ITS2 ribosomal DNA was extracted and amplified using polymerase chain reaction (PCR) methods. The PCR products were then sequenced and analyzed based on GenBank information obtained. An analysis by Restriction Fragment Length Polymorphism using ITS2 PCR products on An. punctipennis was conducted. Seven hundred ninety samples were processed to look at the identity and geographic distribution of Holarctic Anophlines. An. puntipennis has no current ITS2 records in GenBank. The distribution of An. daciae and An. persiensis was analyzed by ITS2 and location data. The identity and presence of a malaria vector in new areas or existing areas would prove to be vital if the disease were to re-emerge due to climate changes. / Master of Science in Life Sciences / Diseases like malaria claims the lives of millions of people every year. This deadly disease can result in considerable morbidity which presently affects countries in Africa, Eurasia, and South America. Anopheline mosquitoes transmit this disease. African Anophelines are very well studied species however, there has been no recent significant research for Holarctic Anophelines, in particular in the United States because of the eradication of malaria in the northern territories and the focus on other diseases transmitted by Aedes and Culex mosquitoes. One of the understudied neglected malaria vectors that has an extensive geographic distribution throughout the United States is Anopheles punctipennis. This species can transmit both forms of human malaria Plasmodium falciparum and Plasmodium vivax. Accurate morphological or molecular identification of mosquitoes is important for proper surveillance, control and diagnostic measures. Identifying this mosquito on a molecular level is pivotal for future genomic research in identifying vector competence, and insecticide resistant genes associated with this species. Internal Transcribe Spacer 2 sequencing is an efficient molecular tool for the identification of Anopheline mosquitoes. This tool has been successfully developed for species from the Maculipennis group and A. crucians complex but not for An. punctipennis type species. The goal of this study was to develop simple and robust molecular tools for identifying this species in the fields. Anopheline mosquito collections were made from multiple locations in the Mid-west to eastern U.S. Sequencing on the ribosomal DNA internal transcribed spacer 2 region (ITS2) of the genome provides positive and accurate identification of An. punctipennis. Developing a Restriction Fragment Length Polymorphism (RFLP) assay using the ITS2 PCR products reduces time and cost in molecular identification and proves to be an accurate method of identification. This research will enable future population genetic studies that are important for the development of mosquito population control.