A maximum-likelihood method for the estimation of tetrad frequencies from single-spore data is presented. The multilocus exchange with interference and viability (MEIV) model incorporates a clearly defined model of exchange, interference, and viability whose parameters define a multinomial distribution for single-spore data. Maximum-likelihood analysis of the MEIV model (MEIVLA) allows point estimation of tetrad frequencies and determination of confidence intervals. We employ MEIVLA to determine tetrad frequencies among 15 X chromosomes sampled at random from Drosophila melanogaster natural populations in Africa and North America. Significant variation in the frequency of nonexchange, or E(0) tetrads, is observed within both natural populations. Because most nondisjunction arises from E(0) tetrads, this observation is quite unexpected given both the prevalence and the deleterious consequences of nondisjunction in D. melanogaster. Use of MEIVLA is also demonstrated by reanalyzing a recently published human chromosome 21 dataset. Analysis of simulated datasets demonstrates that MEIVLA is superior to previous methods of tetrad frequency estimation and is particularly well suited to analyze samples where the E(0) tetrad frequency is low and sample sizes are small, conditions likely to be met in most samples from human populations. We discuss the implications of our analysis for determining whether an achiasmate system exists in humans to ensure the proper segregation of E(0) tetrads.