Three molecular genetic techniques, restriction fragment length polymorphisms (RFLPs), random amplification of polymorphic DNA (RAPD), and allozyme variability, were used to evaluate the genetic diversity of two specific-pathogen-free (SPF) populations (numbers 1 and 2) and one candidate SPF population (number 4) of Penaeus vannamei developed and maintained by the U.S. Marine Shrimp Farming Program. A total of 114 individuals were tested, which included 30 each from families 1.5 and 1.6 of population 1 and from population 2, and 24 from population 4. Two HhaI mitochondrial DNA polymorphisms (A and B) were found in all the animals examined, with family 1.5 and population 2 showing type A and family 1.6 showing type B. After scoring 73 bands obtained with six different RAPD primers, the percentage of polymorphic bands was: 55% for families 1.5 and 1.6 of population 1, 48% for population 2, and 77% for population 4, suggesting that population 4 is the most polymorphic of all three populations. The allozymic variation at 30 loci showed no fixed differences in isozyme genotypes between families 1.5 and 1.6. The percentage of polymorphic loci, under the criterion that the frequency of the most common allele was less than 0.95 in each population, was 6.67%, 3.33% and 16.67% for family 1.5 of population 1, family 1.6 of population 1, and population 2, respectively. Mean heterozygosities (+/- SE) were 0.023 +/- 0.017, 0.018 +/- 0.016, and 0.064 +/- 0.026, respectively. The low levels of allozyme polymorphisms indicate that mitochondrial DNA and nuclear DNA techniques are more useful for examining genetic diversity in order to follow individual stocks within a breeding program and to correlate genotypes with desirable growth and reproductive performance of SPF P. vannamei stocks.