A method for labeling synthetic oligonucleotide probes to high specific activity is described. The method utilizes two partly complementary oligonucleotides that are labeled by a fill-in reaction using the Klenow fragment of DNA polymerase I and four alpha 32P-nucleoside triphosphates. Such probes can, in combination with Southern blot analysis, be used for routine analysis of individual genes in multigene families.