DNA polymorphisms were detected in Homalodisca coagulata (Say) (Homoptera: Cicadellidae) with the following DNA fingerprinting methods: inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) and primer pair-ISSR-PCR (pp-ISSR-PCR), randomly amplified microsatellite polymophisms (RAMP), selective amplification of microsatellite polymorphic loci (SAMPL), and primer pair-random amplification of polymorphic DNA-polymerase chain reaction (pp-RAPD-PCR). But first, a small-scale DNA fingerprinting screening procedure was initiated with these methods with a few individual insects to estimate the most sensitive and efficient method(s). In total, 205 polymorphic markers were generated with the four methods. The efficiency ratio estimated the following order for each method: 1) pp-ISSR-PCR and ISSR-PCR, 2) RAMP, 3) pp-RAPD-PCR, and 4) SAMPL. The screening efficiency ratio estimated that pp-ISSR-PCR and ISSR-PCR were the most efficient methods. DNA polymorphisms were detected in a natural population of 10–30 insects. The number of polymorphic loci ranged from five (pp-RAPD-PCR reaction 6) to 32 (ISSR-PCR primer 13), and the percentage of polymorphic loci was 100% for most primers tested. DNA fingerprinting methods tested were able to detect geographic variation in populations of H. coagulata from Bakersfield and Riverside, CA, and Weslaco, TX. Dendrograms based on Nei’s genetic distance showed that H. coagulata from Bakersfield and Riverside formed a cluster separate from Weslaco in three DNA fingerprinting reactions tested incorporating simple sequence repeats. DNA fingerprinting methods tested were also able to distinguish between three Homalodisca sharpshooters: H. coagulata, Homalodisca insolita (Walker), and Homalodisca liturata (Ball). The present results confirmed the utility of the DNA fingerprinting screening procedure and demonstrated, for the first time, genetic variation in natural populations of glassy-winged sharpshooters by PCR-based DNA fingerprinting methods.