It has been shown that alpha-glyoxal and its derivatives possess antivirus and antitumor activities. Eighteen new coumarin 3-glyoxal derivatives were synthesized in our laboratory. The fragmentation pattern of MS and the characteristic signals of 1HNMR of these compounds have also been studied. In pharmacological test in vitro most of these analogues showed antimutagenic activities, among them, compound 9 exhibited very strong antimutagenic activity and eight compounds showed strong effects. The struture-activity relationship and the possible active substructure responsible for the activity of these compounds were discussed. As expected, coumarin 3-glyoxals showed higher antimutagenic activities than their 3-acetyl coumarin counterparts. We also found that alkylation or esterification of 7-hydroxy were favorable to their activities.