OBJECTIVE: Wound healing can be delayed by the presence of colonising bacteria, and in polymicrobial wounds they may act synergistically to the further detriment of wound healing. In this pilot investigation, biopsy and swab samples were obtained as part of skin-graft operations performed on a chronic venous leg ulcer in order to study the spatial microbial diversity and to compare standard bacteriological and molecular biological techniques. METHOD: The wound was sampled before excision, and sampling was undertaken at multiple locations across the wound. Swab samples and biopsies were subjected to culture analysis and 16S rRNA polymerase chain reaction (PCR), and to denaturing gradient gel electrophoresis (DGGE). RESULTS: Within the wound samples, DGGE identified the major wound microflora components and established the extent of local differences in bacterial diversity. CONCLUSION: This ongoing investigation has verified DGGE as a powerful tool for elucidating the clinical microbiology of a chronic disease state. It also suggests that skin graft operations are a novel way of obtaining multiple samples for in vivo bacteriology and for establishing the spatial distribution of bacteria in the complex micro-environment of chronic wounds.