Micro-exons are a kind of exons with lengths no more than 51 nucleotides. They are generally ignored in genome annotation due to the short length, whereas recent studies indicate that they have special splicing properties and important functions. Considering that there has been no genome-wide study of micro-exons in plants up to now, we screened and analyzed genes containing micro-exons in two indica rice varieties in this study. According to the annotation of Zhenshan 97 (ZS97) and Minghui 63 (MH63), ~23% of genes possess micro-exons. We then identified micro-exons from RNA-seq data and found that >65% micro-exons had been annotated and most of novel micro-exons were located in gene regions. About 60% micro-exons were constitutively spliced, and the others were alternatively spliced in different tissues. Besides, we observed that approximately 54% of genes harboring micro-exons tended to be ancient genes, and 13% were Oryza genus-specific. Micro-exon genes were highly conserved in Oryza genus with consistent domains. In particular, the predicted protein structures showed that alternative splicing of in-frame micro-exons led to a local structural recombination, which might affect some core structure of domains, and alternative splicing of frame-shifting micro-exons usually resulted in premature termination of translation by introducing a stop codon or missing functional domains. Overall, our study provided the genome-wide distribution, evolutionary conservation, and potential functions of micro-exons in rice.