This study examines the biodiversity of mushrooms collected from Bowie State University campus, USA and in Enugu State, Nigeria. Mushroom specimens, meticulously collected and documented, were employed to fulfill the objective of characterizing mushroom diversity. Utilizing the DNA Subway’s phylogenetic analysis tool, DNA barcodes were analyzed, and phylogenetic trees were constructed to compare data. The findings of this investigation have the potential to deepen our understanding of local mushroom communities and provide valuable insights into the similarities among mushroom species found in Nigeria and Maryland. Notably, the results unveiled a genetic similarity spectrum of 59%-99% in Nigeria and 57%-99% in Bowie State University, Maryland. Moreover, distinct clustering of samples from Bowie State University and Nigeria was observed in the constructed phylogenetic tree.