Context. Brassica juncea germplasm exhibits significant variations in flowering timing and vernalisation requirements. However, knowledge gaps exist with respect to variations in expression and the divergent evolution of flowering genes in B. juncea subgenomes.

Aims. This study aims to examine the role of flowering genes in defining trait variation and to identify indications of directional selection on these genes.

Methods. Employing a combination of genome-wide association studies, functional genomics and population genetic assays, we explored the genetic architecture underlying flowering time variation within expansive germplasm collections of this allopolyploid and its progenitor species.

Key results. Genome-wide association studies aided in predicting 17 and 34 candidate genes in B. rapa and B. juncea, respectively. Seven of these (FT, FLC, BAG4, ELF4-L2, EFM, SEP4, and LSH6) were predicted in both B. juncea and B. rapa. Some genes, GA20OX3, NF-YA1, PI, MMP, RPS10B, CRY2, AGL72, LFY, TOC1, ELF5, EFM, FLC and TFL1 exhibited directional selection as inferred from negative Tajima’s D and Fu’s Fs statistics.

Conclusions. Common predicted genes are known influencers of flowering time and phenological changes between species as well as across zones of adaptation. An analysis of gene expression patterns indicates that the gene expression bias in resynthesised B. juncea could be influenced by the cytoplasmic background. Most expression variants are found in B genome copies. Some genes lacked expression variation in their diploid progenitors, whereas these genes exhibit expression variation in polyploid species.

Implications. This study highlights that integrating genome-wide association studies with molecular signals of natural selection can effectively contribute to our understanding of the ecological genetics of adaptive evolution.