The climbing cucurbit Echinocystis lobata, native to North America and alien to many European countries, was assessed for its genetic diversity and differentiation across its introduced range of populations by applying markers of amplified fragment length polymorphism (AFLP). Various tests, including an evaluation of the intrapopulation diversity, principal coordinate, and molecular variance analyses, showed that the Central and Eastern European populations differing in geography and arrival history are also distinct in the genetic parameters. Genetic diversity, defined as the percentage of polymorphic AFLP loci, ranged within 28–62% (on average 51%) at the regional scale (in Romanian, Baltic State, and Central Russian populations), and was very similar to this parameter at the local scale (on average 52% for Lithuanian populations). The differentiation was significant among the populations of the regions (Ф = 0.125, p = 0.001) and at the local scale (among the Lithuanian populations of the different river basins, Ф = 0.058, p = 0.010). The Bayesian results suggested the presence of three genetic clusters among the 29 sites, with populations from Romania, Latvia, Estonia, and the northern part of Lithuania comprising one prevailing cluster, populations from the Nemunas river basin of Lithuania comprising either the former mentioned cluster or the second cluster, and populations of Central Russia comprising the third genetic cluster. Overall, E. lobata in Europe has probably originated from multiple introductions. The intentional anthropogenic seed dispersal by marketing accompanied by hydrochory might have an impact on such a profile of genetic clusters.