Introduction Potato (Solanum tuberosum) belongs to the family Solanaceae and is an important agricultural crop. It is the world's fourth largest food crop, following rice, wheat, and maize. Distributed worldwide with about 5000 varieties, the potato has its center of origin in the high Andes of South America. It is best known for its carbohydrate content in the form of starch, though it also contains important vitamins and minerals (Abbas et al., 2012). It plays a viable role in the worldwide sustainable food system, producing more food energy on less land with low cost of cultivation. It is the most important tuber crop in terms of production, accounting for about 45% of total world tuber crop production (Shewry, 2003) and parental marker studies in anther-derived progeny (Aziz, 2014a, 2014b). MicroRNAs (miRNAs) are single-stranded RNAs that are noncoding, endogenous, and 18 to 26 nt in length (Bartel, 2004). The intergenic genes of miRNAs are generated from endogenous stem-loop primary transcripts known as precursor miRNAs (pre-miRNAs) through actions of Dicer-like 1 (DCL1), an RNAse III enzyme. DCL1, along with other proteins, generates a mature miRNA from the cleavage of stem-looped pre-miRNA (Ambros, 2003). Mature miRNA further associates with argonaute proteins to develop an RNA-induced silencing complex (RISC). The RISC then functions as a guide molecule in the negative posttranscriptional gene, silencing through 2-way base pairing with target mRNAs. The base pairings are perfect or near perfect and lead to mRNA cleavage or translational repression, respectively. miRNAs are one of the largest gene families, accounting for approximately 1% of the genome (Bartel, 2004). Many recent studies have shown that miRNAs have vital roles in diverse regulatory pathways in plants and animals (Baloch and Din, 2014; Din and Barozai, 2014a). In plants, miRNAs regulate tissue differentiation and development, including leaf, root, stem, and flower tissues. They also regulate developmental timing from the vegetative to reproductive stages, as well as signal transduction and response to biotic and abiotic stresses, such as salinity, drought, cold, heat, and pathogens (Zhang et al., 2006; Baloch et al., 2013; Song et al., 2013). The miRNAs and their targets interact through complex regulatory networks. A single miRNA can bind to regulate several distinct mRNA targets and, conversely, Abstract: MicroRNAs (miRNAs) are tiny, endogenous, nonprotein-coding, and functionally negative regulators of posttranscriptional gene regulation. Potato (Solanum tuberosum L.) is a starchy and tuberous crop that is also important for its potassium and vitamin contents. Though potato is the world's fourth largest food crop, its miRNAs and their targets have not been well studied. Here we report a comparative genomics approach that was applied to explore miRNAs and their targeted proteins in potato. A total of 120 new miRNAs from 110 families were identified and characterized from the expressed sequence tags. All 120 miRNAs were observed, along wit...