Anopheles species composition, abundance, and Plasmodium natural infection are important aspects to assess malaria transmission risk. In this study, these aspects were evaluated in a large number of localities in the most important malaria-endemic regions of Colombia. Mosquitoes were collected from 2012 to 2015 in 55 localities of northwestern and western Colombia. Anopheles species composition, abundance, and Plasmodium infection were estimated. A total of 13,218 Anopheles specimens were evaluated. The highest species richness was detected in the northwest, where the main vectors, An. albimanus (27.8%) and An. nuneztovari (26.7%), were the most abundant species. In the west, An. nuneztovari predominated (51.6%), followed by An. darlingi (29.2%). Six species were infected with Plasmodium, An. darlingi, An. nuneztovari, An. albimanus, An. calderoni, An. triannulatus, and An. braziliensis. Results showed that in these localities the main Colombian vectors are widely spread, which suggests a high malaria risk. Notably, this study is the first to report An. braziliensis from Colombia as being naturally infected with Plasmodium. Infection results for species that are suspected local vectors indicate the importance of conducting further studies to assess their epidemiological importance. This information provides the basis for the application of directed vector control strategies that are species-specific.