Abstract Background There is a lack of understanding the evolutionary forces driving niche segregation of closely related organisms. In addition, pinpointing the genes driving ecological divergence is a key goal in molecular ecology. Here, larval transcriptome sequences obtained by next-generation-sequencing are used to address these issues in a morphologically cryptic sister species pair of non-biting midges ( Chironomus riparius and C. piger ). Results More than eight thousand orthologous open reading frames were screened for interspecific divergence and intraspecific polymorphisms. Despite a small mean sequence divergence of 1.53% between the sister species, 25.1% of 18,115 observed amino acid substitutions were inferred by α statistics to be driven by positive selection. Applying McDonald-Kreitman tests to 715 alignments of gene orthologues identified eleven (1.5%) genes driven by positive selection. Conclusions Three candidate genes were identified as potentially responsible for the observed niche segregation concerning nitrite concentration, habitat temperature and water conductivity. Additionally, signs of positive selection in the hydrogen sulfide detoxification pathway were detected, providing a new plausible hypothesis for the species’ ecological differentiation. Finally, a divergently selected, nuclear encoded mitochondrial ribosomal protein may contribute to reproductive isolation due to cytonuclear coevolution.