Soybean mosaic virus (SMV) causes significant yield losses and seed-quality deterioration in the soybean (Glycine max (L.) Merr.) growing areas of China, and breeding disease-resistant cultivars is the most common approach for controlling the spread of the disease and the destruction of soybean crop. In this study, 97 widely grown soybean cultivars representing nine decades (1923–2006) of breeding from the four main soybean-producing subregions in China (Northern Heilongjiang (NH), Mid-Southern Heilongjiang (MSH), Jilin-Liaoning (JL) and Yellow–Huai-Hai River Valleys (YHH)) were inoculated with six prevalent SMV strains: SC3, SC7, SC8, SC11, SC15 and SC18. The average disease index (ADI) of the six SMV strains ranged from 26.95 to 48.97, and the numbers of resistant and susceptible cultivars to the six SMV strains ranged from 27 (27.8%) to 64 (66.0%) and 33 (34.0%) to 70 (72.2%), respectively. The ADIs of cultivars from NH, MSH, JL and YHH were 50.82, 47.27, 43.10 and 33.05, respectively. Soybean cultivars released in the 1940s and 1960s had the highest and lowest ADI values, 53.95 and 32.03, respectively. From NH and JL, all individual strain disease index (DI) values exhibited decreasing trend over time, but no decreasing trend in DI values was observed from MSH. From YHH, DI values for SC3 and SC18 displayed apparent increasing trend over time, and DI values for SC15 showed an obvious decreasing trend. In all, 24 soybean cultivars were identified as having broad-spectrum resistance, with ADI values ranging from 0.80 to 35.52 for the six SMV strains, and 13 soybean cultivars were identified as highly resistant to at least one SMV strain. The findings of this study will contribute to monitoring the pattern of spatio-temporal variation in SMV resistance in different soybean-producing areas of China and facilitate conventional and molecular breeding programs for SMV resistance in soybean.