Recent collection data indicate that at least four potential malaria vectors occupy more widespread distributions within the Andean highlands than in the past. Since habitat elimination is an important aspect of malaria control, it is vital to characterize larval habitats for Anopheles species within both lowland and highland sites. To that end, 276 sites within Ecuador were surveyed between 2008 and 2010. Characteristics of Anopheles-present sites for four species were compared to Anopheles-absent sites within the same geographical range and also to Anopheles-absent sites within a highland range representing potential future habitats. Thermochron iButtons© were used to describe the daily temperature variation within a subset of potential habitats. Anopheles albimanus (W.) was positively associated with permanent habitats, sand substrates, floating algae (cyanobacterial mats), and warmer temperatures in both comparisons. Anopheles pseudopunctipennis (T.) was associated with floating algae (cyanobacterial mats), warmer temperatures, and higher water clarity in both comparisons. Anopheles punctimacula (D.&K.) was negatively associated with floating algae and positively associated with dissolved oxygen in both comparisons. Anopheles oswaldoi s. l. (P.) was not significantly associated with any parameters more often than expected given larval-absent sites. The results indicate that minimum water temperatures might limit the upper altitudinal distribution of An. albimanus (18.7° C) and An. pseudopunctipennis (16.0° C).