Endophytes are microorganisms residing inside plants and are mostly transmitted by seeds. In this study, we used internal transcribed spacer metagenomic sequencing analysis to investigate the fungal endophyte profile of seeds harvested from three different soybean genotypes (OT 13-08, AC Proteus, and AC Harmony) grown in Manitoba, Canada. Protein and oil contents of these genotypes were different from each other. Out of the three soybean genotypes tested, AC Harmony showed the highest level (71.85%) of predicted fungal endophyte taxonomic units classified up to the genus level. At the species level analysis, 20 predicted microorganisms were common in all genotypes. In addition, AC Harmony included the most diverse potential number (90) of endophyte species. The potential roles of the identified endophytes were further studied and the differential roles were observed based on previous evidence. This study will give insight into fungal endophytes in Canadian soybean genotypes for potential applications in agriculture.