Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plantArabidopsis thalianaencodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present inA. thaliananuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins fromA. thaliana, and from two additional Brassicaceae species,Arabidopsis lyrataandBrassica oleracea(cauliflower), fail to bind single-strand telomeric DNAin vitrounder the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts fromB. oleracea, partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities inA. thalianaand its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.