Three independent genetic loci have been shown to contribute to soybean Glycine max (L.) Merrill seed linolenic acid levels, including the well-characterized Fan locus. Linolenic acid is the product of omega-3 fatty-acid desaturase enzyme activity. The objective of this study was to identify and characterize the family of soybean omega-3 fatty-acid desaturase genes and link them to low seed linolenic acid as a tool for the development of molecular markers for low linolenic acid soybean. Using database homology searches and gene cloning, we identified and characterized three soybean microsomal omega-3 fatty-acid desaturase genes that contribute to seed linolenic acid levels. Relative expression was characterized by quantitative real-time RT-PCR (reverse transcriptase-polymerase chain reaction). One of the three genes was predominantly expressed in developing seeds. We determined that the low linolenic acid breeding line A5 (fan fan) contains two of the genes, but is missing the third sequence. Therefore, the Fan locus can be definitively assigned to one of the three microsomal omega-3 fatty-acid desaturase genes present in the soybean genome. Molecular markers for defects in the three genes will enhance soybean programs breeding for low linolenic acid.