Genome walking is a basic molecular biology technique for obtaining unknown sequences. In biology, unknown sequences are usually related to gene regulation, diseases, undiscovered functions of genes, and food safety. The first genome walking method described, inverse PCR (I-PCR), has been established for 28 years. Despite the shortcomings of I-PCR, it has permitted the identification of several previously unknown sequences. To overcome the drawbacks of I-PCR and enhance its efficiency, and in conjunction with recent advances in molecular biology, many genome walking methods have been developed. Essentially, these methods may be classified into the two following categories: those with or those without genome digestion involving restriction enzymes in the first step. According to the features of specific genome walking techniques, these methods are suitable for different types of templates, such as long or short genome walking, single or multiple insertion identification, or simple or complex DNA structures. As the world begins to place greater importance on food security, the future of genome walking is promising, especially in identifying the unintended consequences of GM foods.