MicroRNAs (miRNAs) are a type of interfering RNAs which are small, similar to 22 nucleotides long and remain evolutionary conserved. They have been reported in a large number of plants including other organisms like viruses, insects and higher vertebrates where they have biological and metabolic activities. Their pivotal role in gene silencing has been correlated with a number of diseases; therefore miRNA prediction is an important and challenging field. In spite of being a significant medicinal plant, no specific work has been done yet on the microRNAs (miRNAs) and their potential targets in Cannabis sativa. In vitro prediction of miRNAs is a slow and laborious process as it is hard to isolate them. In our current study a well-developed, faster and powerful in silico approach, EST-based homology search is followed to identify potential miRNA of C. sativa. Here, the Expressed Sequence Tag (EST) database of Cannabis sativa was BLASTed against the previously identified plant miRNAs and following a series of filtration criteria, a total of 2 potential miRNAs were identified. Furthermore, 14 potential mRNA targets were tagged in C. sativa genome based on the complementarity between miRNAs and their targeted mRNA sequences. The predicted target genes encode mainly transcriptional regulators including R2R3-Myb transcription factor, GRAS family transcription factor, Scarecrow protein, Transcriptional regulator family protein and DNA binding protein. Some other targets have more specific functions in the developmental process of C. sativa and are associated with diverse metabolic processes for stress management as well as other metabolic functions. These include Leucine rich protein kinase family protein, Retro element pol polyprotein, Pectin acetyl Esterase, SET domain protein, HXXXD-type acyl-transferase family protein, Ankyrin repeat family protein, Hypersensitivity related protein. Overall, findings from our study will pave the way for further researches on C. sativa miRNAs and their functions. (C) 2016 Elsevier B.V. All rights reserved.