Osedax worms are whale-fall specialists that infiltrate whale bones with their root tissues. These are filled with endosymbiotic bacteria hypothesized to provide their hosts with nutrition by extracting organic compounds from the whale bones. We investigated the diversity and distribution of symbiotic bacteria in Osedax mucofloris from shallow-water whale-falls in the North Atlantic using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH). We observed a higher diversity of endosymbionts than previously described from other Osedax species. Endosymbiont sequences fell into eight phylogenetically distinct clusters (with 91.4–98.9% similarity between clusters), and considerable microdiversity within clusters (99.5–99.7% similarity) was observed. Statistical tests revealed a highly significant effect of the host individual on endosymbiont diversity and distribution, with 68% of the variability between clusters and 40% of the variability within clusters explained by this effect. FISH analyses showed that most host individuals were dominated by endosymbionts from a single cluster, with endosymbionts from less abundant clusters generally confined to peripheral root tissues. The observed diversity and distribution patterns indicate that the endosymbionts are transmitted horizontally from the environment with repeated infection events occurring as the host root tissues grow into the whale bones.