RNA N-methylguanosine (m2G) is one kind of posttranscriptional modification and plays crucial roles in the control and stabilization of tRNA. However, our knowledge about the biological functions of m2G is still limited. The key step of revealing its new function is to recognize the m2G sites in the transcriptome. Since there is no effective method for detecting m2G sites, it is desirable to develop new methods to identify m2G sites. In this study, a computational predictor called iRNA-m2G was proposed to identify m2G sites in eukaryotic transcriptomes. In iRNA-m2G, the RNA sequences were encoded by using nucleotide chemical property and accumulated nucleotide frequency. iRNA-m2G was not only validated by the rigorous jackknife test on the benchmark dataset but also examined by performing cross-species validations. In addition, iRNA-m2G was also tested on an independent dataset. It was found that the accuracies obtained by iRNA-m2G were all quite promising in these tests, indicating that the proposed method could become a powerful tool for identifying m2G sites. Finally, a user-friendly web server for iRNA-m2G is freely accessible at http://lin-group.cn/server/iRNA-m2G.php.