Oxytricha trifallax is a ciliated protozoa located in the freshwater lakes and streams of Indiana. It contains a genetic organization including two separate nuclei, the micronucleus and macronucleus. These two nuclei separate the genetic functions of the cell between them. One highly characterized region of the genome for the trifallax is the 81 gene locus, but it has only been studied in two strains of this species. In order to broaden understanding of this region, the 81 locus was sequenced for 21 natural isolates to better understand the allelic variation in natural populations. All the strains were made up of homozygous or heterozygous combinations of the previously identified 3, A, and B alleles. There were only new variations of identified alleles previously identified in natural strains, and there were no identified natural triallelics. The lack of new alleles and level of homozygosity in their naturally isolated strains suggests that this species consists of isolated subpopulations with little interbreeding between these groups.