Aim To determine the identity and diversity of endophytes in soybean plants using culture-dependent (CD) and culture-independent (CI) methods. Methods and Results Stem samples were collected from three field-grown soybean cultivars grown to a reproductive stage in Minnesota, USA. Samples were surface disinfested, and CD and CI methods were used to assess the endophytes. For the CD method, fungi were isolated and grouped based on colony morphology, and the rDNA ITS region was sequenced to identify the cultures. The most frequently isolated genera were Cladosporium (36%), Alternaria (13%), Diaporthe (9%) and Epicoccum (9%). For the CI method, DNA was extracted from the stems, and the ITS region was amplified, cloned and sequenced for identification. The most prevalent genus detected using CI method was Cladosporium (85%). Conclusions Soybean contains a diverse array of endophytic fungi that were identified in this study. The CD method detected greater endophyte diversity (H'=2 center dot 12) than the CI method (H'=0 center dot 66). Significance and Impact of the Study The results improve our understanding of the identity and diversity of endophytic fungi that likely have different kinds of interactions with soybean plants. The results suggest that CD and CI methods should be used to study endophytes in soybean and perhaps other annual crop plants.