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The <i>Knotted1-like homeobox</i> (<i>KNOX</i>) gene family plays a pivotal role in regulating meristem activity, organ differentiation, and cell meristematic identity. However, there has been a lack of large-scale, systematic, and comprehensive comparative analyses to explore their expression patterns and evolutionary mechanisms. In this study, a total of 1425 <i>KNOX</i> genes were identified across 118 plant species. The result showed that higher plants exhibited a significantly higher abundance of <i>KNOX</i> genes compared to lower plants. Phylogenetic analysis revealed that all <i>KNOX</i> genes can be divided into two classes (class I and II) and evolved independently after species differentiation. An analysis of gene duplication or loss showed that gene loss was more common than gene duplication in lower plants within the <i>KNOX</i> gene family. These findings suggest that gene loss in the <i>KNOX</i> gene family occurs after events such as whole-genome duplication (WGD) or whole-genome triplication (WGT). In addition, conserved motif analysis was also conducted to uncover the evolutionary trajectories of <i>KNOX</i> genes. We found that three motifs (M1, M2, and M4) were present in nearly all <i>KNOX</i> genes, while four novel motifs (M7–M10) were lost in lower plants but present in higher plants. Moreover, the loss of certain motifs in the <i>KNOX</i> genes was also observed in higher plants, indicating sequence divergence in <i>KNOX</i> genes throughout evolution. To understand the expression patterns of <i>KNOX</i> genes, a gene expression pattern analysis was performed in <i>A. thaliana</i> and <i>O. sativa</i>. The results showed that class I <i>KNOX</i> genes exhibit conserved high expression in stems, suggesting their potential similar biological roles across different plant species and the conservation of their functions during evolution. Additionally, we analyzed the <i>KNOX</i> genes in the Citrus genus and closely related species, and we found that the number of <i>KNOX</i> genes evolved at a slower rate in these species, indicating a relatively conservative nature. In conclusion, this study provides valuable resources for the investigation of <i>KNOX</i> gene family evolution and function in plants.