As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.