BACKGROUND: Phosphorus (P) is an essential macronutrient for Brassica napus L. growth and development, and is mainly acquired from the soil as phosphate (Pi). However, there is no research on the system analysis of Pi utilization related genes (PURs) in B. napus yet. RESULTS: In this study, 285 PURs were identified in B. napus genome, including 4 transcription factor (TF) gene families (83 genes) and 17 structural gene families (202 genes). Subcellular localization analysis showed that the proteins encoded by B. napus PURs were mainly located in the nucleus (~ 46.0%) and cell membrane (~ 36.5%). Chromosome localization analysis suggested that B. napus PURs were distributed on An (131) and Cn (149) subgenomes without bias. Analysis of 35 representative species confirmed that PURs were widely present in plants ranging from Chlorophyta to angiosperms with a rapid expansion trend. Collinearity analysis revealed that allopolyploidization and small-scale duplication events resulted in the large expansion of B. napus PURs. For each gene pair of B. napus PURs, the sequence identity of promoter was significantly lower than that of CDS, proving the significant difference in promoter region that might be related to the divergence of PURs expression and function. Transcription factor (TF) binding site prediction, cis-element analysis, and microRNA prediction suggested that the expressions of B. napus PURs are regulated by multiple factors including 32 TF gene families (362), 108 types of CRE (29,770) and 25 types of miRNAs (66). Spatiotemporal expression analysis demonstrated that B. napus PURs were widely expressed during the whole developmental stages, and most synteny-gene pairs (76.42%) shared conserved expression patterns. RNA-seq analyses revealed that most B. napus PURs were induced by low Pi stress, and the hub genes were generally the Pi transporter (PHT) family members. qRT-PCR analysis proved that the expression levels of four B. napus PURs were positively correlated with the root system architecture of three B. napus varieties under low Pi supply at the seedling stage. CONCLUSION: The 285 PURs were identified from B. napus with strong LP inducible expression profile. Our findings regarding the evolution, transcriptional regulation, and expression of B. napus PURs provide valuable information for further functional research.