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Background: Several recent climate change reviews have stressed the possibility of some malaria vectors occupying regions of higher altitudes than previously recorded. Indeed, highland malaria has been observed in several African nations, possibly attributable to changes in land use, vector control and local climate. This study attempts to expand the current knowledge of the distribution of common Anopheles species in Ecuador, with particular attention to highland regions (> 500 m) of the Andes., Methods: Extensive field collections of larvae were undertaken in 2008, 2009 and 2010 throughout all regions of Ecuador (except the lower-altitude Amazonian plain) and compared to historical distribution maps reproduced from the 1940s. Larvae were identified using both a morphological key and sequencing of the 800 bp region of the CO1 mitochondrial gene. In addition, spatial statistics (Getis-Ord Hotspot Analysis: Gi*) were used to determine high and low-density clusters of each species in Ecuador., Results: Distributions have been updated for five species of Anopheles in Ecuador: Anopheles albimanus, Anopheles pseudopunctipennis, Anopheles punctimacula, Anopheles eiseni and Anopheles oswaldoi s.l.. Historical maps indicate that An. pseudopunctipennis used to be widespread in highland Andean valleys, while other species were completely restricted to lowland areas. By comparison, updated maps for the other four collected species show higher maximum elevations and/or more widespread distributions in highland regions than previously recorded. Gi* analysis determined some highland hot spots for An. albimanus, but only cold spots for all other species., Conclusions: This study documents the establishment of multiple anopheline species in high altitude regions of Ecuador, often in areas where malaria eradication programs are not focused.