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The microRNAs are endogenous, regulating gene expression either at the DNA or RNA level. Despite the availability of extensive studies on microRNA generation in plants, reports on their abundance, biogenesis, and consequent gene regulation in plant organelles remain naVve. Building on previous studies involving pre-miRNA sequencing in Abelmoschus esculentus, we demonstrated that three putative microRNAs were raised from the chloroplast genome. In the current study, we have characterized the genesis of these three microRNAs through a combination of bioinformatics and experimental approaches. The gene sequence for a miRNA, designated as AecpmiRNA1 (A. esculentus chloroplast miRNA), is potentially located in both the genomic DNA, i.e., nuclear and chloroplast genome. In contrast, the gene sequences for the other two miRNAs (AecpmiRNA2 and AecpmiRNA3) are exclusively present in the chloroplast genome. Target prediction revealed many potential mRNAs as targets for AecpmiRNAs. Further analysis using 5N RACE-PCR determined the AecpmiRNA3 binding and cleavage site at the photosystem II protein N (psbN). These results indicate that AecpmiRNAs are generated from the chloroplast genome, possessing the potential to regulate mRNAs arising from chloroplast gene(s). On the other side, the possibility of nuclear genome-derived mRNA regulation by AecpmiRNAs cannot be ruled out. [ABSTRACT FROM AUTHOR]