Fusarium graminearum is the primary pathogen responsible for Fusarium Head Blight (FHB) in cereals, causing yield losses and mycotoxin contamination. The present study investigated the genetic diversity and population structure of F. graminearum from wheat grains and maize residues, which were the primary inoculum sources of FHB. A total of 588 isolates from four different fields were genotyped using a sequence-based microsatellite genotyping method of 34 SSR markers (SSRseq) and characterized for chemotype distribution. The results revealed high genetic diversity with 453 distinct multilocus genotypes (MLGs) without significant genetic differentiation by geographic location or substrate, suggesting a single metapopulation at the regional scale. This absence of substrate genetic differentiation strongly supports the fact that residue-borne inoculum contributed to FHB infection, although we failed to demonstrate any field-specific dispersal pattern from residues to grains. The findings underscore the importance of maize residues as a critical inoculum source and highlight the need to consider both local and regional sources in FHB management.