Endophytes associated with crops have potential as beneficial inoculants in agriculture, but little is known about their genetic diversity and phylogenetic relationships. We carried out the first ever ecological and phylogenetic survey of the culturable fungal root endophytes of a wild barley species. Fungal root endophytes were isolated from 10 populations of wall barley (Hordeum murinum), and 112 taxa of fungi were identified based on internal transcribed spacer sequence similarity. We found representatives from 8 orders, 12 families and 18 genera. Within this group, only 34 isolates (30% of the total) could be confidently assigned to a species, and 23 of the isolates (21% of the total) had no significant match to anything deposited in GenBank (based on <85% sequence similarity). These results suggest a high proportion of novel fungi, with 28% not assigned to a known fungal order. This includes three endophytes that have been shown to significantly improve agronomic traits in cultivated barley. This study has, therefore, revealed a profound diversity of fungal root endophytes in a single wild relative of barley. Extrapolating from this, the study highlights the largely unknown, hugely diverse and potentially useful resource of crop wild relative endophytes.