The molecular genetic changes underlying the transformation of wild plants into agricultural weeds are poorly understood. Here we use a sunflower cDNA microarray to detect variation in gene expression between two wild (non-weedy) Helianthus annuus populations from Utah and Kansas and four weedy H. annuus populations collected from agricultural fields in Utah, Kansas, Indiana, and California. When grown in a common growth chamber environment, populations differed substantially in their gene expression patterns, indicating extensive genetic differentiation. Overall, 165 uni-genes, representing approximately 5% of total genes on the array, showed significant differential expression in one or more weedy populations when compared to both wild populations. This subset of genes is enriched for abiotic/biotic stimulus and stress response proteins, which may underlie niche transitions from the natural sites to agricultural fields for H. annuus. However, only a small proportion of the differentially expressed genes overlapped in multiple wild vs. weedy comparisons, indicating that most of the observed expression changes are due to local adaptation or neutral processes, as opposed to parallel genotypic adaptation to agricultural fields. These results are consistent with an earlier phylogeographic study suggesting that weedy sunflowers have evolved multiple times in different regions of the United States and further indicate that the evolution of weedy sunflowers has been accompanied by substantial gene expression divergence in different weedy populations.