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MiR396s play important roles in regulating plant growth and stress response, and great potential for crop yield promotion was anticipated. For more comprehensive and precise understanding of miR396s in Poaceae, we analyzed the phylogenetic linkage, gene expression, and chromosomal distribution of miR396s in this study. Although the mature miR396s' sequences were mostly conserved, differential expression patterns and chromosomal distribution were found among Poaceae species including the major cereal crops rice, wheat, and maize. Consistently, in comparison with rice, wheat and maize plants transformed with the target mimicry construct of miR396 (MIM396) exhibited differential effects on grain size and disease resistance. While the TaMIM396 plants showed increased grain size, panicle length and sensitivity to B. graminis , the ZmMIM396 plants didn't show obvious changes in grain size and disease resistance. In Addition, several GROWTH-REGULATING FACTOR (GRF) genes in wheat and maize were repressed by miR396s, which could be reversed by MIM396, confirming the conserved regulatory roles of miR396 on GRFs. While providing new solution to enhance grain yield in wheat and revealing potential regulatory variations of miR396s in controlling grain size and disease resistance in different crops, this study gives clues to further explore miR396s' functions in other Poaceae species. • Poaceae miR396s are divided into three groups and display both conserved and diversified evolutionary characterizations. • Inhibition of miR396s results in increased grain size, panicle length and pathogen sensitivity in wheat but not in maize. • Different GRFs were upregulated in the MIM396 transgenic plants compared to their corresponding wild type in wheat and maize. [ABSTRACT FROM AUTHOR]