An important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species, different methodologies for the determination of insertion sites using a range of published protocols and commercially available kits were assessed in transgenic lines of varying degrees of complexity, from low copy number to complex re-transformed and co-transformed lines. Three commercial kits, APAgene™ GOLD Genome Walking Kit (BIO S&T), DNA Walking SpeedUp™ Kit II (Seegene), and Universal Vectorette™ System (Sigma) were compared with an adaptor-mediated PCR technique. Overall, the APAgene™ kit was used with a high success rate with low copy number potato lines, and also more complex co- and re-transformed lines, and adhering to key confirmation steps it was possible to obtain flanking sequence ranging in size from 300 to 2,500 bp and eliminate PCR artefacts from the analysis.