Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidiosis, an infectious diarrheal disease primarily affecting humans and neonatal ruminants. Understanding the transmission dynamics of C. parvum, particularly the specific contributions of zoonotic and anthroponotic transmission, is critical to the control of this pathogen. This study used a population genetics approach to better understand the transmission of C. parvum in the Upper Midwest United States. A total of 254 C. parvum isolates from cases of human cryptosporidiosis in Minnesota and Wisconsin and diarrheic calves in Minnesota, Wisconsin, and North Dakota were genotyped at eight polymorphic loci. Isolates with a complete profile from all eight loci (n = 212) were used to derive a multilocus genotype (MLT), which was used in population genetic analyses. Among the 94 MLTs identified, 60 were represented by a single isolate. Approximately 20% of isolates belonged to MLT 2, a group that included both human and cattle isolates. Population analyses revealed a predominantly panmictic population with no apparent geographic or host substructuring.