Fatty acid desaturases can introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In the present study, 29 full-length desaturase genes were identified from soybean genome by a thorough annotation exercise. A comprehensive analysis was performed to characterize phylogeny, chromosomal locations, structures, conserved motifs, and expression patterns of those genes. The soybean genes were phylogenetically clustered into nine subfamilies with the Arabidopsis counterparts, FAB2, FAD2, FAD3, FAD5, FAD6, FAD7, FAD8, SLD1, and DES1. Twenty-nine desaturase genes were found to be distributed on at least 15 of the 20 soybean chromosomes. The gene structures and motif compositions were considerably conserved among the subfamilies. The majority of desaturase genes showed specific temporal and spatial expression patterns across different tissues and developmental stages based on microarray data analyses. The study may provide new insights into the origin and evolution of fatty acid biosynthesis pathways in higher plants. Additionally, the characterization of desaturases from soybean will lead to the identification of additional genes for genetic modification of plants to produce nutritionally important fatty acids.