Accumulation of information about natural hybridization between GM soybean (Glycine max) and wild soybean (Glycine soja) is required for risk assessment evaluation and to establish biosafety regulations in Japan. This is particularly important in areas where wild relatives of cultivated soybean are grown (i.e. East Asia including Japan). To collect information on temporal and spatial factors affecting variation in hybridization between wild and GM soybean, a two year hybridization experiment was established that included one wild soybean and five GM soybean cultivars with different maturity dates. Hybridization frequencies ranged from 0 to 0.097%. The maximum hybridization frequency (0.097%) was obtained from wild soybean crossed with GM soybean cv. AG6702RR, which were adjacently cultivated with wild soybean, with 25 hybrids out of 25 741 seedlings tested. Cultivar AG6702RR had the most synchronous flowering period with wild soybean. Ten hybrids out of 25 741 were produced by crossing with cv. AG5905RR, which had the second most synchronous flowering period with wild soybean. Most hybrids were found where GM and wild soybeans were adjacently cultivated, whereas only one hybrid was detected from wild soybean plants at 2 m, 4 m and 6 m from a pollen source (GM soybean). Differences in flowering phenology, isolation distance and presence of buffer plants accounted for half of the variation in hybridization frequency in this study. Temporal and spatial isolation will be effective strategies to minimize hybridization between GM and wild soybean.