The cosmopolitan freshwater oligochaete Tubifex tubifex is an important health indicator of the aquatic environment. Water pollutants can affect the intraspecific biodiversity grade of local Tubifex colonies. A genetic profile of specimens collected in an environment with reduced anthropic presence is particularly important to define genetic variability in unperturbed Tubifex populations, and it is still missing in international literature. Therefore it has been performed the analysis of lineage composition in a T. tubifex colony from high mountain spring ponds in Central Italy, characterized by a very low concentration of heavy metals. The sampling was performed during spring 2010 and 2011, in the Sett’acque valley (1900 m amsl), within the township of Lucoli. Data presented in this work depicted a peculiar composition of the population, characterized by a reduced complexity compared to other urban environments, and with no specimens belonging to the lineage I, largely described in many European populations. Interestingly lineage 2e, previously discovered only in a natural reserve, results to be the most common lineage in this population. Considerations on environmental health and genetic evolution are discussed.