Carcinoma of the uterine cervix is one of the most common malignancies worldwide, yet it is clearly preventable by population screening. The Papanicolaou (Pap) smear has proved to be the most successful test for the detection of precancerous lesions and is largely responsible for the reduction of cervical cancer mortality and morbidity rates. However, the Pap smear is not perfect; false-negative results of various rates are reported. To improve the diagnostic efficacy of cervical cytology, we performed microsatellite analysis on paired Pap smear samples from cervical lesions. Nine microsatellite markers were chosen from chromosomal regions commonly displaying loss of heterozygostity (LOH) in cervical cancer and those displaying microsatellite instability (MI) in other squamous cell cancer. Microsatellite alterations were detected in 16/21 (76%) Pap smear DNA samples including 11 of 13 (85%) smears from invasive squamous cell carcinomas (SCCs) and 5 of 8 (63%) from squamous intraepithelial lesions (SILs). Microsatellite alterations detected in the Pap smear DNA were identical to those identified in seven paired primary tumors available for analysis. Moreover, this molecular approach detected genetic alterations in two cases apparently negative by cytologic examination. None (0/25) of the control patients displayed microsatellite alterations in paired Pap smears. Microsatellite analysis of cervical cytologic samples may provide a complementary method to analyze suspicious but not diagnostic cytologic samples further.