在分子生物学中，限制性片段长度多态性(restriction fragment length polymorphism, RFLP)具有两种涵义：一是DNA分子由于核苷酸串行的不同而产生的一种可以用来相互区别的性质；二是一种实验技术，利用这种性质来比较不同的DNA分子。这种技术可以用于遗传指纹和亲子鉴定。

In molecular biology, restriction fragment length polymorphism, or RFLP, is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces and (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. RFLP analysis was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing.