Plant chlorophyll content and chlorophyll A:B ratios are influenced by genotype as well as numerous environmental conditions. Due to its importance to primary production, chlorophyll has been studied extensively. While a range of comparative studies between species and among a few genotypes of a range of species have been conducted, no large scale examination of the chlorophyll A:B ratio has been conducted for the soybean germplasm. The objective of this study was to survey the chlorophyll A:B ratios of >800 lines from the USDA soybean germplasm collection. Leaf disks collected from fully light-exposed mature leaves were extracted in 96% ethanol and chlorophyll A and B concentrations were determined using a spectrofluorometer. The examined genotypes separated into two groups in respect to their chlorophyll A:B ratios: one group containing the majority of the examined genotypes had an average chlorophyll A:B ratio of 3.8, and the second group had an average of 2.6. Maturity group (MGV and VI were included) did not appear to affect the chlorophyll A:B ratio. The explanation for the separation into the two distinct groups is not clear but is currently being examined. Ecophysiological implications of these findings warrant further investigations and may be of interest for soybean breeders and agronomists.