Wheat is one of the most important staple crops worldwide, and an important source of human protein and mineral element intake. Continuously increasing stable production of wheat is critical for global food security under the challenge of population growth and limited resource input. Spike architecture determines the potential grain yield of wheat. However, the mechanisms of transcriptional regulation of spike architecture in wheat remain largely unknown, limiting further genetic improvement of wheat yield. In this study we explored the genetic basis of spike architecture in wheat. Population RNA-seq methods were used to identify the eQTLs and sQTLs associated with spike architecture and applied this to dissection of the genetic basis of gene expression and splicing controlling these complex yield-related traits. In total, 4,143 expression quantitative trait loci (eQTLs) and 12,933 splice QTLs (sQTLs) were identified in wheat based on 178 RNA-seq samples, revealing 774 cis-eQTLs and 321 cis-sQTLs for 86 eGenes and 73 sGenes, respectively. Integration of eQTLs and sQTLs with genome-wide association study (GWAS) identified dozens of additional novel candidate genes that may contribute to spike-related traits. Gene network analysis showed that eQTLs and sQTLs were widely involved in the co-expression modules that regulate wheat spike architecture. Notably, the eQTL locus AX-108754757 regulated the expression of 5 eGenes that negatively controled grain number per spike. AX-111592099 regulated both the splicing and expression of TraesCS7B02G442100, encoding an E3 ubiquitin ligase, and playing a central role in regulating spike length. This study provides new insights into the genetic basis of spike architecture. This improved understanding of spike-related traits in wheat will contribute to more rapid genetic improvement.