The architecture of wheat (Triticum aestivum) inflorescence and its complexity is among the most important agronomic traits that influence yield. For example, wheat spikes vary considerably in the number of spikelets, which are specialized reproductive branches, and the number of florets, which are spikelet branches that produce seeds. The large and repetitive nature of the three homologous and highly similar subgenomes of wheat has impeded attempts at using genetic approaches to uncover beneficial alleles that can be utilized for yield improvement. Using a population-associative transcriptomic approach, we analyzed the transcriptomes of developing spikes in 90 wheat lines comprising 74 landrace and 16 elite varieties and correlated expression with variations in spike complexity traits. In combination with coexpression network analysis, we inferred the identities of genes related to spike complexity. Importantly, further experimental studies identified regulatory genes whose expression is associated with and influences spike complexity. The associative transcriptomic approach utilized in this study allows rapid identification of the genetic basis of important agronomic traits in crops with complex genomes.Grains of cereal crops provide a major source of human diet and nutrition. Improving grain yield is a primary objective during crop domestication and a major goal of crop-breeding programs. Inflorescence (spike) architecture dictates the capacity for seed production in cereal crops, including wheat (Triticum aestivum), the world's most widely grown cereal. In the archetypal bread wheat spike, the inflorescence meristem forms a limited number of lateral spikelet meristems (SMs) per rachis node, and a single terminal SM at the distal end. Each SM is indeterminate and typically produces two to four fertile florets that produce seeds (Fig. 1A; Supplemental Fig. S1; Bonnett, 1936;Fisher, 1973). Like maize (Zea mays) and rice (Oryza sativa), wheat yield per plant largely depends on the number of florets per spike and thus spike architecture. The numbers of SMs (and rachises) and florets per spikelet are major target traits for efforts aimed at improving wheat yield. In addition, the number of SMs per rachis may be increased to increase floret number, as in rare supernumerary spikelet variations.In rice, maize, and barley (Hordeum vulgare), multiple genes regulating spike development have been identified (Sreenivasulu and Schnurbusch, 2012;Tanaka et al., 2013). However, our understanding of wheat spike development remains rudimentary at the molecular level. In wheat, increased transcription levels of Q, an AP2-like gene, was markedly associated with spike compactness, suggesting that the Q gene may be implicated in spike development (Simons et al., 2006), although its role in spike complexity remains unclear. Feng et al. (2017) underlying the genetic regulation of rare supernumerary spikelet variations. For example, wheat Photoperiod1 (Ppd1) was identified as a regulator of paired spikelet formation (Boden et al., 2...