In this study, the composition and structure of dipterans of the Alvarado River basin were characterized along with some physicochemical parameters of the area. Between September (dry season) and December (rainy season) of 2012, Diptera in 9 sampling stations in the Alvarado River and some of its tributaries between altitudes of 351 and 1057 m a.s.l. were examined. Biological material was collected across different substrates (sand, leaf litter, gravel and rock) with a Surber net. A total of 3283 larvae belonging to 8 families and 41 genera were collected, including new records for 20 genera in the Tolima region. Chironomidae, Ceratopogonidae and Simuliidae families exhibited the highest density and abundance. September (dry season) reported greater densities, richness and abundances and higher values of diversity and the stations Alvarado-Inicio, Quebrada Chumba and Quebrada la Caima had the highest values of abundance. Also, water samples were collected for physicochemical and bacteriological analyses to determine relationships with dipteran biota. Electrical conductivity, pH, total dissolved solids, alkalinity and hardness had a positive influence on the distribution of aquatic dipteran larvae of the Alvarado River basin.