The conservation and diversity of microRNA (miRNA) families provide insights into the evolution of miRNA genes. However, there are few studies to explore the miRNA genes at the genus level in plants. Here, we identified 1194 miRNA loci in four Populus species P. deltoides, P. euphratica, P. tremula, and P. trichocarpa from Aigeiros, Turanga, Populus, and Tacamahaca sections, respectively, by combining de novo and homolog-based approaches. Our results indicated that a similar number of miRNA loci exist in each species (296–301 miRNA loci). Among the identified 143 miRNA families, 68 families are shared by the studied four species, and 31 families are species-specific, which might be related to local adaptation. Additionally, multiple miRNA-related single nucleotide polymorphisms (SNPs) were found, indicating that polymorphisms in pre-miRNA hairpins were likely to affect miRNA biogenesis. This study expanded the breadth and depth of miRNA annotations and provided valuable resources for further exploring the diversity and function of poplar miRNAs.