Previously, the genes encoding non-specific lipid transfer proteins (nsLTPs) of the Poaceae family appear to evidence different genomic distribution and somewhat different shares of EST clones, which is suggestive of independent duplication(s) followed by functional diversity. To further evaluate the evolutionary fate of the Poaceae nsLTP genes, we have identified Ka/Ks values, conserved, mutated or lost cis-regulatory elements, responses to several elicitors, genome-wide expression profiles, and nsLTP gene-coexpression networks of both (or either) wheat and rice. The Ka/Ks values within each group and between groups appeared to be similar, but not identical, in both species. The conserved cis-regulatory elements, e.g. the RY repeat (CATGCA) element related to ABA regulation in group A, might be reflected in some degree of long-term conservation in transcriptional regulation post-dating speciation. In group A, wheat nsLTP genes, with the exception of TaLTP4, evidenced responses similar to those of plant elicitors; however, the rice nsLTP genes evidenced differences in expression profiles, even though the genes of both species have undergone purifying selection, thereby suggesting their independent functional diversity. The expression profiles of rice nsLTP genes with a microarray dataset of 155 gene expression omnibus sample (GSM) plates suggest that subfunctionalization is not the sole mechanism inherent to the evolutionary history of nsLTP genes but may, rather, function in concert with other mechanism(s). As inferred by the nsLTP gene-coexpression networks, the functional diversity of nsLTP genes appears not to be randomized, but rather to be specialized in the direction of specific biological processes over evolutionary time.