Nine lines of T2 generation of marker-flee transgenic soybeans of Pudou 8008 with △6-fatty acid desaturase gene were characterized. PCR and Southern blot indicated that the selective marker gene (bar) had been removed from some transgenic soybean progenies, while △6-fatty acid desaturase gene was still in some progenies of these transgenic soybean lines. Northern blot showed that the △6-fatty acid desaturase gene could be transcribed in the marker-free transgenic soybean lines. Gas chromatographic analysis showed that six transgenic soybean lines could produce γ-linolenic acid.