The importance of microRNAs (miRNAs) at the post-transcriptional regulation level has recently been recognized in both animals and plants. In this study, the simple and most effective method of comparative genomic approach was used. First known plants miRNAs BLAST against the soybean genome, and then the located candidates were searched for novel miRNAs by RNA folding method in the vicinity (±400 nt) of the candidates. The results showed that a total of 521 novel soybean miRNA genes, including 236 mature miRNAs, were identified. All these mature miRNAs were grouped into 58 families, of which 21 of them were novel family in soybean. The upstream 2 000 nt of potential pre-miRNAs was used for promoter prediction, in order to investigate prediction of miRNAs and detect transcript unit and clustering. In this study, miRNA genes less tend to be present as clusters in soybean. Only 9 clusters, containing 21 miRNA genes (accounted for 4.0% of the total), were observed as part of polycistronic transcripts. Detailed analysis of sequence characteristics of novel miRNAs in soybean and all previous known plants miRNAs, were carried out. These results of this study provide a reference point for further study on miRNAs identification in plants, and improve the understanding of genome in soybean. The importance of microRNAs (miRNAs) at the post-transcriptional regulation level has recently been recognized in both animals and plants. In this study, the simple and most effective method of comparative genomic approach was used. First known plants miRNAs BLAST against the soybean genome , and then the then candidates were searched for novel miRNAs by RNA folding method in the vicinity (± 400 nt) of the candidates. The results showed that a total of 521 novel soybean miRNA genes, including 236 mature miRNAs, were identified. All these The upstream 2 000 nt of potential pre-miRNAs was used for promoter prediction, in order to investigate prediction of miRNAs and detect transcript unit and clustering. In this study, miRNA genes less tend to be present as clusters in soybean. Only 9 clusters, containing 21 miRNA genes (accounted for 4.0% of the total), were observed as part of polycistronic transcripts. Detailed analysis of sequence characteristics of novel miRNAs in soybean and all previous known plants miRNAs, were carried out.