Positive selection and relaxation of purifying constraints after duplication events have driven the functional diversification of gene families involved in development. One example of this occurred within the plant MADS-box genes. The evolution of the orchid flower was driven by duplication events followed by sub- and neo-functionalization of class B DEF-like MADS-box genes, which are present at three to four copies in the orchid genome. In contrast, the orchid PI/ GLO-like class B MADS-box genes have been reported thus far as single-copy loci, with the only exception of Habenaria radiata. We isolated a novel PI/ GLO-like gene ( OrcPI2) in Orchis italica, which is different than the previously characterized OrcPI locus. The presence of two functional paralogs of PI/ GLO-like genes in orchids is detectable only within the tribe Orchidinae. Evolutionary analyses revealed an apparent relaxation of purifying selection acting on the two PI/ GLO-like paralogs of the Orchidinae when compared to the single-copy PI/ GLO-like genes found in other orchid species. Furthermore, by measuring d N/d S ( ω) ratios, we show that a high percentage of sites between the two PI/ GLO-like paralogs have different evolutionary pressures. Interestingly, the apparent relaxation of selective constraints on the two PI/ GLO-like paralogs is due to strong purifying selection at synonymous sites rather than to a high value of nonsynonymous substitution rate. This peculiar evolutionary pattern might be related to molecular processes such as mRNA folding and/or translational efficiency control. These processes could potentially be involved in or predate the functional diversification of the two PI/ GLO-like paralogs within Orchidinae.