MicroRNAs are a ~22 nucleotide small non-coding RNAs found in animals, plants and viruses. They regulate key cellular processes by enhancing, degrading or silencing protein coding targets. Currently most of the data on miRNA is available from Drosophila. Given their important post-transcriptional role in several organisms, there is a need to understand the miRNA mediated processes in normal and abnormal conditions. Here we report four novel microRNAs ast-mir-2502, ast-mir-2559, ast-mir-3868 and ast-mir-9891 in Anopheles stephensi identified from a set of 3,052 transcriptome sequences, showing average minimum free energy of −31.8 kcal/mol of duplex formation with mRNA indicating their functional relevance. Phylogenetic study shows conservation of sequence signatures within the Class Insecta. Furthermore, 26 potential targets of these four miRNAs have been predicted that play an important role in the mosquito life-cycle. This work leads to novel leads and experimental possibilities for improved understanding of gene regulatory processes in mosquito. © 2015, Springer Science+Business Media Dordrecht.