Tiller development in low-tillering wheat is related to several differentially expressed genes, proteins, and metabolites, as determined by an integrated omics approach combining transcriptome analysis, iTRAQ, and HPLC-MS on multiple NILs. Tillering is an important aspect of plant morphology that affects spike number, thereby contributing to the final crop yield. However, the mechanisms inhibiting tiller production in low-tillering wheat are poorly characterized. To investigate this aspect of wheat biology, two pairs of near-isogenic lines were developed, and an integrated omics approach combining transcriptome analysis, isobaric tags for relative and absolute quantification, and high-performance liquid chromatography-mass spectrometry were used to compare the free-tillering and low-tillering caused by an allele at Qltn.sicau-2D in wheat samples. Overall, 474 genes, 166 proteins, and 28 metabolites were identified as tillering-associated differentially expressed genes, proteins, and metabolites (DEGs, DEPs, and DEMs, respectively). Functional analysis indicated that the abundance of DEGs/DEPs/DEMs was related to lignin and cellulose metabolism, cell division, cell cycle processes, and glycerophospholipid metabolism; three transcription factor families, GRAS, GRF, and REV, might be related to the decrease in tillering in low-tillering wheat. These findings contribute to improve our understanding of the mechanisms responsible for the inhibition of tiller development in low-tillering wheat cultivars.