Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.