Wheat (Triticum aestivum L.) is one of the most important crops in the world. Four spike-related traits, namely, spike weight (SW), spike length (SL), the total number of spikelets per spike (TSNS), total kernels per spike (TKNS), and thousand-kernel weight (TKW), were evaluated in 270 F3:6 Nebraska winter wheat lines in two environments (Lincoln and North Platte, NE, USA). All genotypes in both locations exhibited high genetic variation for all yield traits. High positive correlations were observed among all yield-related traits in each location separately. No or low correlation in yield-related traits was observed between the two environments. The broad-sense heritability estimates were 72.6, 72.3, 71.2, 72.3, and 56.1% for SW, SL, TSNS, TKNS, and TKW, respectively. A genome-wide association study (GWAS) was used to identify SNPs associated with yield traits. In the Lincoln environment, 44 markers were found to be significantly associated with spike-related traits (SW, SL, TSNS, TKNS, and TKW), while 41 were detected in North Platte. Due to the strong significant genotype x environment, no common SNP markers were found between the two locations. Gene annotation of the significant markers revealed candidate genes encoded for important proteins that are associated directly or indirectly with yield traits. Such high genetic variation among genotypes is very useful for selection to improve yield traits in each location separately.