Abstract Key message The vacuolar processing enzyme gene TaVPE3cB is identified as a candidate gene for a QTL of wheat pith-thickness on chromosome 3B by BSR-seq and differential expression analyses. Abstract The high pith-thickness (PT) of the wheat stem could greatly enhance stem mechanical strength, especially the basal internodes which support the heavier upper part, such as upper stems, leaves and spikes. A QTL for PT in wheat was previously discovered on 3BL in a double haploid population of ‘Westonia’ × ‘Kauz’. Here, a bulked segregant RNA-seq analysis was applied to identify candidate genes and develop associated SNP markers for PT. In this study, we aimed at screening differentially expressed genes (DEGs) and SNPs in the 3BL QTL interval. Sixteen DEGs were obtained based on BSR-seq and differential expression analyses. Twenty-four high-probability SNPs in eight genes were identified by comparing the allelic polymorphism in mRNA sequences between the high PT and low PT samples. Among them, six genes were confirmed to be associated with PT by qRT-PCR and sequencing. A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential PT candidate gene in Australian wheat ‘Westonia’. A robust SNP marker associated with TaVPE3cB was developed, which can assist in the introgression of TaVPE3cB.b in wheat breeding programs. In addition, we also discussed the function of other DEGs which may be related to pith development and programmed cell death (PCD). A five-level hierarchical regulation mechanism of stem pith PCD in wheat was proposed.