Rhizobia seed inoculants are commonly used by soybean [Glycine max L. (Merr.)] producers in the Midwest to promote adequate root nodulation and nitrogen fixation for the growing season. However, studies suggest that this is unnecessary in crop rotations which frequently incorporate soybean production. The objective of this study is to develop an accurate method for quantifying soybean-associated soil rhizobia by means of quantitative polymerase chain reaction (qPCR). Such a technique could provide growers with timely seed treatment recommendations and allow for further study of rhizobia populations within various cropping systems. Rhizobia were quantified with the qPCR method and compared to the most probable number (MPN) according to plant infection rates. A standard curve was developed using liquid cultures with a population gradient of Bradyrhizobium japonicom USDA110, and then verified using soil samples from a long term corn (Zea mays)/soybean rotation study. Primers for qPCR were selected based upon the nodZ and noeI specificity genes present in Bradyrhizobium japonicum USDA 110, Bradyrhizobium elkanii USDA 76, Rhizobium etli CFN 42 and Sinorhizobium fredii.