Abstract The objective of this study was to detect the expression profile of microRNAs (miRNAs) in two breeds of fat-tailed sheep (Guangling Large-tailed sheep and Small-tailed Han sheep), and discover the miRNAs that may play roles in lipid metabolism. Consequently, two small RNA libraries were constructed and high-throughput sequencing was used to identify the miRNAs expressed in the two libraries and to calculate the expression of miRNAs. Bioinformatics methods were used to predict target genes, which were subsequently analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG). Nine miRNAs (miR-10b, miR-29a, miR-30c, miR-155, miR-192, miR-206, novel-miR-102, novel-miR-36, and novel-miR-63) were chosen to verify the sequencing results by detecting their expressions in the two breeds using quantitative real-time PCR. A total of 113 and 131 conserved miRNAs in the two breeds were obtained, of which 40 miRNAs were differentially expressed. KEGG analysis showed that many predicted target genes for these miRNAs are involved in lipid metabolism pathways. By screening the unknown miRNAs using MIREAP, 208 and 215 novel miRNAs were obtained from the two libraries, of which 150 were significantly different. This study represents the comprehensive survey of miRNAs expressed in the tail fat of two breeds of sheep with different tail fat deposition using HiSeq analysis. We conclude that differentially expressed miRNAs may play roles in regulating adipose metabolism, and thus, may contribute to improving the meat quality and fat deposition in sheep.