The rifampicin resistance mutations induced by low energy nitrogen ion implantation had been investigated in this study referred to the study of 60Co-gamma ray irradiation. It is suggested that low energy nitrogen ion beam implantation might generate a lower damage but get higher mutation rates than 60Co-gamma ray irradiation did. The frequencies showed the preference of base substitutions induced by N+ ion beam are CG--> TA transition, GC-->AT transition, AT--> GC transition and AT-->TA transversion which occupy 87.5% (77/88) of the total mutation of base substitutions. Two new rif-determining sites had also been identified in rpoB gene in Escherichia coli. The first new site located in 1551. When dG1551 was replaced by dC, it resulted in Gln517 was substituted by a histidine. The other new site located in 1692. When dC1692 was replaced by dT, it resulted in a Pro564 is changed into a leucine. The synonymous mutation of the second site had been reported but the nucleotide substitution, dC-->dT, had not been identified before.