We present data on Chironomidae (Insecta: Diptera) collected in South America together with results on the mitochondrial DNA diversity within selected megadiverse genera. This work is part of an on-going project on the ancient origin of South American biodiversity using non-biting midges. Collections were made at 42 localities, in March 2014 and February 2015, in a diverse array of habitats, including small streams, rivers, ponds, lakes and bays. In total, 3196 representatives of six subfamilies were collected. Sixty-one genera were identified, containing at least 211 species. The subfamilies Chironominae and Orthocladiinae predominated in all samples. Tanypodinae were often present, but rarely in large numbers. Except for Podonomus pepinellii, reported from Brazil, Podonominae were collected in a few localities in Argentina (Arroyo Lopez, and Arroyo Gutierrez and Gutierrez Lake) and Chile (Llanquihue Lake). Prodiamesinae were only recorded in Chile. Analysis of DNA barcode sequences using neighbor-joining estimation supported 66 species within the selected genera. The chironomid fauna of South America includes multiple genera with worldwide distributions, with Australian, Nearctic and Neotropical components.