Microsatellite markers are used in genotypic studies of Diabrotica spp. Usually commercial kits are used for DNA extraction from preserved tissues. A simplified procedure, based on the CTAB, was used to obtain DNA from tissues preserved in 75% ethanol and from fresh samples of Diabrotica spp. from Durango and Jalisco, Mexico. Significantly more DNA was obtained from fresh than preserved tissues (926 and 827 ng/µL, respectively). However, the quality of DNA was similar (A260nm/A280nm mean value = 1.93) between the two kinds of tissue. The CTAB procedure was used to isolate similar quality and quantity of DNA as obtained by using commercial kits but was less time consuming and expensive. The DVV-D2 loci, a characteristic microsatellite of Diabrotica genus, was amplified.