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Acyl carrier proteins (ACPs) have been reported to play a crucial role in responding to biotic and abiotic stresses, regulating growth and development. However, the biological function of the <i>ACP</i> gene family in the <i>Brassica</i> genus has been limited until now. In this study, we conducted a comprehensive analysis and identified a total of 120 <i>ACP</i> genes across six species in the <i>Brassica</i> genus. Among these, there were 27, 26, and 30 <i>ACP</i> genes in the allotetraploid <i>B. napus</i>, <i>B. juncea</i>, and <i>B. carinata</i>, respectively, and 14, 13, and 10 <i>ACP</i> genes in the diploid <i>B. rapa</i>, <i>B. oleracea</i>, and <i>B. nigra</i>, respectively. These <i>ACP</i> genes were further classified into six subclades, each containing conserved motifs and domains. Interestingly, the majority of <i>ACP</i> genes exhibited high conservation among the six species, suggesting that the genome evolution and polyploidization processes had relatively minor effects on the <i>ACP</i> gene family. The duplication modes of the six <i>Brassica</i> species were diverse, and the expansion of most <i>ACPs</i> in <i>Brassica</i> occurred primarily through dispersed duplication (DSD) events. Furthermore, most of the <i>ACP</i> genes were under purifying selection during the process of evolution. Subcellular localization experiments demonstrated that <i>ACP</i> genes in <i>Brassica</i> species are localized in chloroplasts and mitochondria. <i>Cis</i>-acting element analysis revealed that most of the <i>ACP</i> genes were associated with various abiotic stresses. Additionally, RNA-seq data revealed differential expression levels of <i>BnaACP</i> genes across various tissues in <i>B. napus</i>, with particularly high expression in seeds and buds. qRT-PCR analysis further indicated that <i>BnaACP</i> genes play a significant role in salt stress tolerance. These findings provide a comprehensive understanding of <i>ACP</i> genes in <i>Brassica</i> plants and will facilitate further functional analysis of these genes.