Plant microRNAs are important endogenous gene regulators which regulate gene expression at post-transcriptional level. Previous studies have identified that miR169 family members regulated the NF-YA transcription factors which have been implicated in plant development and stress responses. At present, reported potato genome sequence data offered an opportunity for global insights into the molecular mechanisms of the miR169/NF-YA modules in potato. In this work, 4 novel stu-miR169 family members were predicted in potato based on potato genome sequence data. miRNA target prediction showed that mature stu-miR169 sequences have a bite sit on the 5 of StNF-YA genes in potato, and three of them were validated by RNA ligase-mediated 5′RACE (5′ RLM-RACE) assay. The result from investigation of the expression patterns of mature stu-miR169 and their predicted target genes also showed that mature stu-miR169 was down-regulated in response to the drought stress. There were some targeted StNF-YA genes that exhibited a negative expression pattern with mature stu-miR169 during the different periods of drought-treated samples. Taken together, the decreased expression of stu-miR169 might drive over-expression of NF-YA family members, they are related to resistances against drought stress.