Pubescence is an important morphological trait in soybean, which plays an important role in growth and development, and which is correlated to many agronomic traits such as 100-seed weight, and deserves investigation. In this study, four soybean mutants with developmental defect in pubescence were selected from EMS mutant library: the mutant ddsp1 with short pubescence, the glabrous mutant ddsp2, the mutant ddsp3 with partially shrunken pubescence, and the mutant ddsp4 with fully shrunken pubescence. Scanning electron microscope images showed that the length and morphology of pubescence were significantly altered in these mutants. In addition, the seed size and 100-seed weight were all significantly different from the wild type. Ten soybean orthologous genes, including GL1, GL2, GL3and TCL1 which act on the initiation of pubescence development, RBR1, SIM, KAK and SPY which participate in negative regulation of endoreduplication, and CPR5 and RHL which participate in positive regulation of endoreduplication, were selected for expression analysis in those four mutants and the wild type by qRT-PCR. The results showed that the genes controlling the initiation of pubescence development, except for GL1 and GL2 which significantly down-regulated the expression in mutants ddsp2 and ddsp4, up-regulated the expression or had no significant effect. The gene RBR1 significantly up-regulated the endoreduplication in mutant ddsp1 only, whereas the genes SIM, KAK and SPY significantly down-regulated the endoreduplication in all the four mutants, and both the genes CPR5 and RHL significantly up-regulated the endoreduplication in all the four mutants. Through the expression analysis of pubescence-related genes in soybean, this research has laid a foundation for further searching for the genes controlling the soybean pubescence development and understanding the regulation mechanisms of these genes.