Three Galium species are believed to be present across western Canada: Galium aparine, Galium spurium and Galium boreale. Galium spurium and G. aparine are very difficult to distinguish morphologically, which is problematic for crop consultants and weed surveyors, and could have implications for control measures. Molecular techniques could potentially make identification easier and more rapid than using chromosome counts, as is currently done. The objective of this study was to identify morphological traits and/or genetic polymorphisms capable of species differentiation. To this end, Galium seed of unknown speciation were collected from nine field populations across western Canada and, along with two reference samples of G. spurium and G. aparine, were characterised for both morphological traits and their ribosomal ITS1-5.8S-ITS2 genomic sequence. In addition, single nucleotide polymorphism variation within the highly conserved 5.8S ribosomal RNA gene was identified that could consistently differentiate Galium species. Sequence analysis of the ITS1-5.8S-ITS2 region of field collections from western Canada indicated that all samples were G. spurium and all were highly related to each other. These results were supported by a distinct lack of variation in morphological traits, as nearly all plant traits measured did not differ between populations. This suggests that all sampled populations, and perhaps most of the Galium populations across western Canada, are derived from a single species, G. spurium.