In 2009 and 2010, isolates of Fusarium oxysporum with translation elongation factor sequences (EF-1α) identical to those of F. oxysporum f. sp. vasinfectum race 4 were found on wilted cotton plants in Alabama and Mississippi. Unlike other races of Fusarium oxysporum f. sp. vasinfectum in the United States, race 4 can cause significant damage to Upland cultivars in the absence of root-knot nematodes; therefore, the discovery of this race in the Southeast could have serious implications. Four of the race 4-like isolates, collected in 2009 and 2010 from Auburn University’s E. V. Smith Research Center in Alabama and Mississippi State University’s R. R. Foil Research Center, were examined further using sequences of the nuclear ribosomal DNA intergenic spacer region (IGS rDNA) in addition to the translation elongation factor. The four southeastern isolates were identical to reference isolates of race 4 in EF-1α sequence, but differed from each other and reference isolates of race 4 in IGS sequence. These results show that EF-1α sequence data alone cannot be used to identify race 4 of F. oxysporum f. sp. vasinfectum, and that the isolates from Alabama and Mississippi are distinct from race 4. Therefore, race 4 of F. oxysporum f. sp. vasinfectum has not yet been found in the southeastern U.S.