In a 2023 survey evaluating conifers with Rosellinia infections, five Pestalotiopsis-like fungal endophytes were isolated from plant samples obtained from Maine, New Hampshire, and Ohio by The Mycology & Nematology Genetic Diversity & Biology Laboratory at the United States Department of Agriculture. The two data sets provided herein contain species-specific base pair substitutions for the partial translation elongation factor 1-alpha gene (TEF). In the alignments, the novel Pestalotiopsis fungi are compared to their most closely related species. The data set can be used as a diagnostic tool to differentiate between the closely related species. DNA was extracted from fungal samples using the E.Z.N.A HP Plant & Fungal DNA Kit (OMEGA® Bio-Tek, Norcross, GA, USA) following manufacturer’s protocol. The TEF locus was amplified, and reactions were conducted in 25 μL volumes with 12.5 μL of KAPA2G Robust Hotstart® (Kapa Biosystems, Inc., Wilmington, MA, USA), 1.25 μL of the forward and reverse primers at 10 μM, 1-1.75 μL of DNA at 10-20 ng, and 8 μL of molecular grade H2O. Amplifications were performed following the protocol described by Maharachchikumbura et al. (2014) in a C1000 Touch PCR Thermal Cycler (Bio-Rad, Hercules, CA). PCR products were analyzed through capillary electrophoresis with the QIAxcel Advanced System instrument and the QIAxcel ScreenGel software (Qiagen, Hilden, Germany). PCR products were then purified using ExoSAP-IT Cleanup (Affymetrix, Santa Clara, CA) following the manufacturer’s protocol. The BigDye™ 3.1 Terminator Cycle sequencing kit was used to sequence amplicons bi-directionally with the Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).Resources in this dataset:Resource Title: TEF P. neohantoniensis alignment, File Name: TEF_P. neohantoniensis alignment, Resource Description: Alignment of TEF DNA sequences for species closely related to Pestalotiopsis neohantoniensis.Resource Title: TEF P. maineana alignment, File Name: TEF_P. maineana alignment, Resource Description: Alignment of TEF DNA sequences for species closely related to Pestalotiopsis maineana.