Abstract: In order to study mosquitoes in North Khorasan Province, northeastern Iran and assess the zoogeographic affinities of the Iranian and Middle Asian fauna, mosquito surveys were conducted in the province during 2005-2009. Adult mosquitoes were collected from resting sites and while landing on human and dog bait by means of aspirator. Larvae were collected by means of pipette and dipper using the standard dipping technique. In total, 1,336 mosquito specimens, including 682 adults and 654 third- and fourth-instar larvae, were collected. Fourteen species representing five genera were identified: Anopheles claviger, An. maculipennis, An. superpictus, An. pulcherrimus, Culex hortensis, Cx. mimeticus*, Cx. modestus*, Cx. perexiguus*, Cx. pipiens*, Cx. theileri, Cx. tritaeniorhynchus, Culiseta longiareolata, Ochlerotatus caspius* and Uranotaenia unguiculata (asterisks indicate new occurrence records for the province) based on morphology and An. maculipennis based on the internal transcribed spacer 2 ribosomal DNA (ITS2 rDNA) sequence. The data show that 65% of the anopheline fauna of Middle Asia occurs in Iran, however at least 15 Anopheles taxa found in Iran are not recorded in Middle Asia. Only seven (28%) of the aedine mosquitoes recorded in Middle Asia are found in Iran and three species that occur in Iran are not found in Middle Asia. About 77% of the Middle Asian species of Culex occur in Iran whereas the Iranian fauna includes nine species that are not recorded in Middle Asia. The species of Coquillettidia, Culiseta and Uranotaenia that are found in Iran are also found in Middle Asia.