MicroRNAs (miRNAs) are a group of small non-coding RNAs that negatively regulate expression of their target genes at the posttranscriptional level. The miRNA-mediated gene regulation has emerged as one of the critical modes of gene regulation important for almost all biological processes of a plant life cycle. However, the role of miRNAs in photosynthesis is unknown. Photosynthesis is a chief metabolic process through which plants synthesize carbohydrates in the presence of sunlight using atmospheric carbon dioxide and water. Flaveria genus belongs to the family Asteraceae and its species differ greatly in their mode of photosynthesis as different Flaveria spp. operate either C3, C3-C4 intermediate, or C4 photosynthesis. This makes Flaveria a valuable model system to investigate, at the molecular level, how C4 type has evolved from the ancestral C3 type. To address whether or not miRNAs have a role in this process, we have analyzed miRNAs in the leaves as well as from the isolated mesophyll and bundle sheath cells of leaves from Flaveria robusta (C3), Flaveria ramosissima (C3-C4 intermediate) and Flaveria bidentis (C4). The analyses revealed significant differences for the abundances of various miRNA families in Flaveria robusta (C3), Flaveria ramosissima (C3-C4 intermediate) and Flaveria bidentis (C4). To gain an insight into the miRNA targets in these plant species, degradome libraries were constructed and sequenced. This approach confirmed targets such as REVOLUTA (member of homeodomain-leucine zipper family) and Target of early activation tagged (EAT) for miR166 and miR172, respectively. Additionally, several other potential targets that could be regulated by miRNAs in the Flaveria spp. have been identified.