Vertically transmitted symbionts associate with some of the most ecologically dominant species on Earth, and their fixation has led to major evolutionary transitions (e.g., mitochondria, chloroplasts). While transmission has been well documented for parasites, for most mutualist symbionts it remains unknown whether vertical transmission is imperfect (symbiont not transmitted to all offspring) in nature and during which host life history stage the symbiont is lost. Through quantitative natural history surveys of fungal endophytes in native grasses, we show that transmission was imperfect for all seven species examined. The type and degree of loss depended on the population and host species, suggesting that transmission varies across geographic mosaics. Our results open new directions for understanding cooperation and conflict in the system. For example, imperfect transmission provides a previously unexplored avenue for host sanctions against costly symbionts. Similarly, endophytes gain opportunities for partner choice that would not exist if transmission were perfect.