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Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of <i>CSE</i> and <i>CSE</i>-like genes in <i>Populus</i>. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of <i>CSE</i> and <i>CSE</i>-like genes, using the poplar (<i>Populus trichocarpa</i>) genome. Eighteen <i>CSE</i> and <i>CSE</i>-like genes were identified in the <i>Populus</i> genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except <i>Oryza sativa</i>. Primary functional classification showed that <i>PoptrCSE1</i> and <i>PoptrCSE2</i> had putative function in lignin biosynthesis. In addition, <i>PoptrCSE2</i>, along with <i>PoptrCSE12</i>, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (<i>Populus tomentosa</i>), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the <i>PoptrCSE</i>-like genes are homologs of <i>AtMAGL</i> (monoacylglycerol lipase) genes in <i>Arabidopsis</i> and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in <i>Populus</i>.