The Fusarium graminearum species complex (FGSC) is an important group of pathogens distributed in maize‐producing areas worldwide. This study investigated the genetic diversity and pathogenicity of 40 FGSC isolates obtained from stalk rot and ear rot samples collected from 42 locations in northeastern China during 2013 and 2014. A phylogenetic tree of translation elongation factor (EF‐la) sequences designated the 40 isolates as F. graminearum sensu stricto (67.5%) and F. boothii (32.5%). By using inter‐simple sequence repeat analysis (ISSR), it was shown that the isolates were divided into two clades, which corresponded to the species identity of the isolates. However, the isolates from the two different diseases could not be distinguished in pathogenicity. The disease severity index of seedlings inoculated with stalk isolates was slightly higher than that of seedlings inoculated with isolates from infected ears, whereas the pathogenicity of the stalk and ear isolates were identical.