Grain yield of wheat is significantly associated with inflorescence or spike architecture. Modifications of spike architecture have been adopted in both early domestication and modern genetic improvement to increase yield. However, few genes related to wheat spike development have been identified and their underlying mechanisms are largely unknown. In this study, we characterized an ethyl methanesulfonate (EMS)-induced wheat mutant, wheat paired spikelet 1 (wps1), with paired spikelet. Unlike single spikelet that usually develops at each node of rachis, the secondary spikelets appeared below the primary spikelets at most of the rachis nodes of wps1. The microscope observation showed that the secondary spikelet initiated later than the primary spikelet did. Genetic analysis suggested that the PS of wps1 is controlled by a single dominant nuclear gene, designated WHEAT PAIRED SPIKELET 1 (WPS1). Further RNA-seq based bulked segregant analysis (BSR) and molecular marker mapping localized WPS1 in an interval of 208.18 Mb-220.92 Mb on the long arm of chromosome 1D, which is different to known genes related to spike development in wheat. By using wheat omics data, TraesCS1D02G155200 encoding a HD-ZIP III transcription factor was considered as the most possible candidate gene for WPS1. Transcriptomic analysis indicated that PS formation in wps1 is associated with auxin-related pathways and may be regulated by networks involving TB1, Ppd1, FT1, and VRN1, etc. This study laid the solid foundation for further validation of the causal gene of WPS1 and explore its regulatory mechanism in PS formation and inflorescence development, which may benefit to kernel yield improvement of wheat based on optimization or design of spike architecture in the future.