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Abstract Six distinct COI mitochondrial Haplotype Groups (HG) are morphologically, ecologically, and genetically characterized from the aquatic nematode family Tobrilidae. Collection locations included the extreme habitats of the Alkaline Lakes in the western Nebraska Sandhills and the contaminated stream, Johnson Creek, bordering the AltEn 2021 catastrophic pesticide release near the village of Mead in eastern Nebraska. Maximum likelihood and genetic distance metrics supported the genetic integrity of the haplotype groups. Discriminant function analysis of COI haplotype group datasets of combined morphological characters and soil chemistry attributes for both male and female Tobrilidae were classified correctly in all but one case. Scanning electron microscopy revealed new details about amphid apertures, male supplements, and spicules. Partial 18S gene phylogeny suggests that the genusSemitobrilusmay not be a member of the subfamily Neotobrilinae, and three specimens in the 226 tobrilid dataset provide evidence of incongruence between COI and 18S derived phylogenies. Given the strong signal provided by the environmental chemistry data, tobrilid mitochondrial haplotypes may well have value as environmental indicators.