Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post-transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue-specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought-responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought-related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress- and tissue development-related genes, including DRP (dehydration-responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD-ZIP (homeodomain-leucine zipper), MYB and NAC-domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought-tolerant tomato breeding.