Phosphate (Pi) transporters play critical roles in Pi acquisition and homeostasis. However, little is known about these transporters in oilseed rape. Therefore, the aim of the present study was to characterize the members of the PHT1 gene family in allotetraploid Brassica napus and to analyze their expression profiles in response to environmental stresses. In total, 49 PHT1 family members were identified in B. napus, including 27 genes in the A subgenome and 22 in the C subgenome. Most of the PHT1 proteins were predicted to localize to the plasma membrane. Phylogenetic analysis suggested that the members of the PHT1 gene family can be divided into seven clades, with the introns/exons and protein motifs conserved in each clade. Collinearity analysis revealed that most of the BnaPHT1 genes shared syntenic relationships with PHT1 members in Arabidopsis thaliana, B. rapa, and B. oleracea, and that whole-genome duplication (polyploidy) played a major driving force for BnaPHT1 evolution in addition to segmental duplication. Transcript abundance analysis showed that a broad range of expression patterns of individual BnaPHT1 genes occurred in response to phosphorus (P) deficiency. In addition, the expression levels of BnaPHT1 genes can be regulated by different nutrient stresses, including nitrogen (N), potassium (K), sulfur (S) and iron (Fe) stresses. Moveover, salt and drought stresses can regulate the transcript abundances of BnaPHT1s, as well as phytohormones including auxin and cytokinin. Gene coexpression analysis based on the RNA-seq data implied that BnaPHT1s might cooperate with each other as well as with other genes to regulate nutrient homeostasis in B. napus. Further analysis of the promoters revealed that GT-1, DRE and P1BS elements are widely distributed within the promoter regions of BnaPHT1 genes. Our results indicate that BnaPHT1s might be involved in cross-talk for sensing the external status of P, N, K, S and Fe, as well as salt and drought stresses. Moreover, these processes might be mediated by phytohormones. Our findings provide the first step in the complex genetic dissection of the Pi transport system in plants and implicate multiple transcriptional regulation, which probably refers to new roles of PHT1 genes in B. napus.