The occurrence and relative abundance of Pythium species were quantified by using a cucumber seed baiting assay and P 10VP agar medium, and compared among three long-term cropping systems: continuous maize (CC), continuous soybean (SS) and maize soybean rotation (CS) at two Iowa locations. Soil types were different at the two locations. Four Pythium species were isolated, P. ultimum, P. torulosum and P. paroecandrum occurred in soils of all three cropping systems, and P. spinosum occurred in one SS and one CS field. More Pythium isolates were recovered from CS and SS composite soil samples than from CC samples. P. torulosum was isolated most frequently.