Abstract <p>Two species of the genus <i>Galinsoga</i>, <i>G. parviflora</i> Cav. and <i>G. quadriradiata</i> Ruiz and Pav., are among the most successful invasive plants causing significant damage to natural- and agroecosystems. Their natural distribution range extends from North to South America, and the adventitious part of the range includes all continents except Antarctica. Despite the practical importance of <i>G. parviflora</i> and <i>G. quadriradiata</i>, the genetic diversity of European populations of these species remains unexplored. In this study, ISSR markers were used to study Ukrainian populations of <i>G. parviflora</i> and <i>G. quadriradiata</i> and compared them with plants from Poland, Lithuania, and Portugal. The results obtained indicate the low genetic diversity (Shannon’s index <i>I</i> = 0.124) of <i>G. quadriradiata</i> populations, which is probably due to the small size of the original population introduced to the Old World from America. In contrast, the genetic diversity in <i>G. parviflora</i> populations is significantly higher (<i>I</i> = 0.254). Some genotypes of <i>G. parviflora</i> have a wide geographical distribution and, at the same time, different genotypes occur in the same area. The data obtained are in good agreement with the hypothesis that the main way of the invasion of <i>Galinsoga</i> species in the Old World was escape from botanical gardens. Among the samples examined, several forms of hybrid nature were identified, probably originating from hybrids between <i>G. parviflora</i> and <i>G. quadriradiata</i>, followed by subsequent backcrossing with one of the parent species.</p>