We have examined how the ILR1-like auxin conjugate hydrolase gene family has functionally evolved in the gymnosperm species Sitka spruce (Picea sitchensis) and Loblolly pine (Pinus taeda). We have isolated and cloned two orthologues from spruce (PsIAR31, PsIAR32) and one from pine (PtIAR31) that are homologous to the Arabidopsis thaliana AtIAR3 auxin amidohydrolase. We have previously examined the enzymatic activity of PsIAR31 using a thin layer chromatography method. Using more sensitive HPLC methods, we have re-investigated the hydrolytic activity and substrate recognition of both PsIAR31 and PsIAR32. Neither PsIAR31 nor PsIAR32 appears to hydrolyze indole butyric acid (IBA) or indole proprionic acid (IPA) conjugates. Additionally, we have found that PsIAR31 and -32 recognize several IAA conjugates (including IAA-ala, IAA-asp, IAA-gluc, and IAA-isoleu). The PtIAR31 enzyme seems to primarily recognize IBA-alanine as a substrate. Using phylogenetic analysis, we also found a family of five putative paralogue hydrolases in both the pine (PtIAR31-35) and the spruce (PsIAR31-35) genomes. This result supports the hypothesis that multiple copies of auxin conjugate hydrolase genes have been present since gymnosperms evolved, and that this redundancy did not arise later in angiosperms.