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MicroRNAs (miRNAs) are small, nonc-oding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development.However, limited information regarding their functions in gymnosperm reproduction is available.Here, we used high-throughput sequencing combined with computational analysis to identify and characterise miRNAs from ovules of Ginkgo biloba, and identified 34 conserved miRNA families and 99 novel miRNAs.The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing.Furthermore, we found that some target genes, e.g.MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF) may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant-pathogen interactions and the biosynthesis of secondary metabolites.Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167,miRNA169, miRNA172 and miRNA390 were upregulated in ovules.Thus, multiple miRNAs closely related to gymnosperm ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.