Plant defensins are small, diverse, cysteine-rich peptides, belonging to a group of pathogenesis-related defense mechanismproteins, which can provide a barrier against a broad range of pathogens. In this study, 51 defensin-like (DEFL) genes inGramineae, including brachypodium, rice, maize and sorghum were identified based on bioinformatics methods. Using thesynteny analysis method, we found that 21 DEFL genes formed 30 pairs of duplicated blocks that have undergone large-scaleduplication events, mostly occurring between species. In particular, some chromosomal regions are highly conserved in thefour grasses. Using mean Ks values, we estimated the approximate time of divergence for each pair of duplicated regionsand found that these regions generally diverged more than 40 million years ago (Mya). Selection pressure analysis showedthat the DEFL gene family is subjected to purifying selection. However, sliding window analysis detected partial regions ofduplicated genes under positive selection. The evolutionary patterns within DEFL gene families among grasses can be used toexplore the subsequent functional divergence of duplicated genes and to further analyse the antimicrobial effects of defensinsduring plant development.