Carotenoids are structurally diverse pigments of biotechnological interest as natural colorants and in the prevention of human disease. The carotenoids present in 19 strains taxonomically related to the poorly described, nonphotosynthetic bacterial genus Hymenobacter, including 10 novel isolates cultivated from Victoria Upper Glacier, Antarctica, were characterized using high-performance liquid chromatography (HPLC). Nine chemically distinct carotenoids, present in various combinations irresolvable by conventional crude spectrophotometric analyses, were purified by preparative HPLC and characterized using UV-visible light absorption spectroscopy and high-resolution mass spectrometry. All major Hymenobacter carotenoids appear to be derived from a common backbone of 2'-hydroxyflexixanthin and include previously unreported presumptive hexosyl, pentosyl, and methyl derivatives. Their distribution does not, however, correlate perfectly with 16S rRNA gene phylogeny. Carotenoid composition, therefore, may be strain specific and does not follow a strictly homogeneous pattern of vertical evolutionary descent.