Heterotrophic N2-fixing bacteria are a potentially important source of N2 fixation in rice fields due to the moist soil conditions. This study was conducted at eight sites along a geographic gradient of the Yangtze River Plain in central China. A nitrogen-free solid malate-sucrose medium was used to isolate heterotrophic N2-fixing bacteria. Numbers of the culturable N2-fixing bacteria expressed as CFU (colony forming units) ranged between 1.410.42106 and 1.240.23108 in the sampled paddy field sites along the plain. Thirty strains with high ARA (acetylene reduction activity) were isolated and purified; ARA of the strains varied from 0.9 to 537.8nmol C2H4 culture-1 h-1, and amounts of 15N fixed ranged between 0.008 and 0.4866mgculture-1day-1. According to morphological and biochemical characteristics, 14 strains were identified as the genus Bacillus, 2 as Burkholderia, 1 as Agrobacterium, 4 as Pseudomonas, 2 as Derxia, 1 as Alcaligenes, 1 as Aeromonas, 2 as Citrobacter, and 3 strains belonged to the corynebacter-form group.