Gene duplication played a fundamental role in eukaryote evolution and different copies of a given gene can be present in extant species, often with expressions and functions differentiated during evolution. We assume that, when such differentiation occurs in a gene copy, this may be indicated by its maintenance in all the derived species. To verify this hypothesis, we compared the histological expression domains of the three β-glucuronidase genes (AtGUS) present inArabidopsis thalianawith the GUS evolutionary tree in angiosperms. We found thatAtGUSgene expression overlaps in the shoot apex, the floral bud and the root hairs. In the root apex,AtGUS3expression differs completely fromAtGUS1andAtGUS2, whose transcripts are present in the root cap meristem and columella, in the staminal cell niche, in the epidermis and in the proximal cortex. Conversely,AtGUS3transcripts are limited to the old border-like cells of calyptra and those found along the protodermal cell line. TheGUSevolutionary tree reveals that the two main clusters (namedGUS1andGUS3) originate from a duplication event predating angiosperm radiation. AtGUS3 belongs to the GUS3 cluster, whileAtGUS1andAtGUS2, which originate from a duplication event that occurred in an ancestor of the Brassicaceae family, are found together in theGUS1cluster. There is another, previously undescribed cluster, called GUS4, originating from a very ancient duplication event. While the copy of GUS4 has been lost in many species, copies of GUS3 and GUS1 have been conserved in all species examined.