Plant microRNAs (miRNAs) play important roles as modulators of gene expression at the post-transcriptional level. Previous studies have shown that high-throughput sequencing is a powerful tool for the identification of miRNAs, and it is believed that many more miRNAs remain to be discovered. Here, we found 23 novel conserved miRNAs from three rice cultivars by high-throughput sequencing and further identified these through subsequent cloning and quantitative real-time polymerase chain reaction (qPCR). Eight of these novel miRNAs were detected with significant signals in the three rice cultivars by northern blotting assays. The quantitative analysis of their expression profiles showed that most of these miRNAs were perfectly or imperfectly negatively correlated with their target genes, which suggests that these miRNAs may play important roles during rice development. This is the first genome-wide investigation of miRNAs from different rice cultivars, and the data obtained expand the known rice miRNA inventory and provide further information about the regulatory roles played by miRNAs in rice development. (C) 2014 Elsevier B.V. All rights reserved.