The potato aphid Macrosiphum euphorbiae (Thomas) is a major pest of several economic crops in Tunisia. Using 10 microsatellites, we analyzed five populations of M. euphorbiae sampled during the 2004–2005 solanaceous and cucurbit season (April through September) from five geographic origins. From 235 aphids, 61 different multilocus genotypes were identified of which three genotypes MLG1, MLG2, and MLG42 were predominant on all host plants and regions. MLG1 and MLG2 genotypes were detected in 2004 and did not reappear in spring 2005, while the genotype MLG42 was detected only in 2005. All populations showed significant deviation from Hardy—Weinberg equilibrium even in data sets including one individual per genotype, χ² independence tests and analysis of molecular variance showed no significant differentiation among populations collected on different host plant and from different geographic origin, but differences between populations from two successive years were significant. Factorial component analysis corroborates these results. The probable causes of this seasonal variation were discussed.