Currently, there is an increasing focus on understanding the interactions between genetic features of the invader and environmental factors that ensure the success of invasion. The objective of our study was to evaluate the genetic diversity of Lithuanian populations of highly invasive small balsam (Impatiens parviflora) by amplified fragment length polymorphism (AFLP) markers and to relate molecular data to biotope features defined by employing neighboring species of herbaceous plants. Low polymorphism of I. parviflora populations was observed at AFLP loci. Hierarchical analysis of molecular variance did not reveal differentiation of populations depending on biotope, geography, or road types. Bayesian analyses of AFLP data demonstrated many genetic clusters. Our results suggest multiple introductions of I. parviflora into Lithuania. The polymorphism of AFLP loci of populations significantly correlated with the total coverage by herbaceous plants in the sites. Defined by principal component analysis, the variability of study sites was most related to the coverage of herbaceous plants and least related to the molecular features of I. parviflora populations. The sites with I. parviflora were classified into agricultural scrubland, riparian forest, and urban forest biotopes. Of them, urban forest was distinguished by the highest coverage of I. parviflora and the lowest Ellenberg indicatory values for light, soil acidity, and richness in nutrients.