Aims: To screen for bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis (DGGE). As members of Bacillus and related genera were found to persist in the final product, this study focussed on these taxa. Methods and Results: Template DNA was extracted from gelatine samples at five crucial points of a gelatine production process. A primer specific for Bacillus and related genera was designed and used in a selective PCR, followed by a nested DGGE-PCR targeting the V9 region of the 16S rDNA. DGGE analysis of the resulting amplicons, and sequence analysis of selected bands, showed high sequence similarities of these bands with Bacillus fumarioli, B. licheniformis, B. coagulans and Clostridium perfringens. When the selective PCR was omitted, primarily Lactobacillus bands were retrieved. Conclusions: PCR-DGGE analysis of gelatine extracts can be used for tracing and screening of bacterial contamination during gelatine production. A selective PCR, nested with DGGE-PCR, gave much more accurate information about endospore-forming contaminants than did the direct DGGE procedure alone. Significance and Impact of the Study: Use of this nested DGGE-PCR protocol may provide important information about possible hazards to the final microbiological quality and/or safety of gelatine, so allowing production parameters and/or remediation procedures may be adjusted on-line.