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Background  Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear.    Results  Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of  Viburnum macrocephalum  f.  keteleeri . We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in  V .  macrocephalum  f.  keteleeri  flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while  SPL  genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be  SPL  genes, we propose that miR156 may be involved in the regulation of stamen development in  V .  macrocephalum  f . keteleeri.Conclusions  We identified miRNAs differentially expressed between fertile and sterile flowers in  V .  macrocephalum  f.  keteleeri  and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.    Electronic supplementary material  The online version of this article (10.1186/s12864-017-4180-x) contains supplementary material, which is available to authorized users.

miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri