We performed miRNA transcriptome analysis of mature medaka tissues using high-throughput next generation sequencing technology. The small RNA libraries were contructed from brain, liver and gonads in mature male and female medaka and a total of more than 128 million sequencing reads were generated from the six small RNA libraries. By comparison to known miRNA database, a total of 223 known miRNA types were identified with more than 50% expressed in brain. The expression profiling showed that almost half of the miRNAs are tissue-specific, with only 55 miRNA types from 34 families common to all tissues. A small number of miRNAs are also gender-specific. The experimental validation was performed for five represented miRNA candidates and four of them are confirmed. Overall design: Investigation of miRNA expression profiles from three tissues (brain, liver and gonads) of mature male and female medaka.