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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 (<i>AtGUS</i>) present in <i>Arabidopsis thaliana</i> with the GUS evolutionary tree in angiosperms. We found that <i>AtGUS</i> gene expression overlaps in the shoot apex, the floral bud and the root hairs. In the root apex, <i>AtGUS3</i> expression differs completely from <i>AtGUS1</i> and <i>AtGUS2</i>, 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, <i>AtGUS3</i> transcripts are limited to the old border-like cells of calyptra and those found along the protodermal cell line. The <i>GUS</i> evolutionary tree reveals that the two main clusters (named <i>GUS1</i> and <i>GUS3</i>) originate from a duplication event predating angiosperm radiation. AtGUS3 belongs to the GUS3 cluster, while <i>AtGUS1</i> and <i>AtGUS2</i>, which originate from a duplication event that occurred in an ancestor of the Brassicaceae family, are found together in the <i>GUS1</i> cluster. 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.