Technical Abstract: Evaluation of past and current biological control programs using molecular tools can clarify establishment success of agent biotypes, and can contribute to our understanding of best practice for natural enemy importations. The flea beetle, Longitarsus jacobaeae has been quite successful at controlling the weed tansy ragwort, Jacobaeae vulgaris, in Pacific coastal areas. The L. jacobaeae biotype introduced in 1969 from Italy is assumed to provide control since establishment of a cold-adapted biotype from Switzerland, also released in 1969 to California, was never confirmed. Recent spread of the weed into Montana to continental, winter-cold climates led to the re-introduction of the Swiss biotype in 2002. The Italian and Swiss biotypes can only be separated by their differing phenologies, and are able to hybridize in the laboratory. We collected 162 L. jacobaeae individuals from 12 populations in the introduced and native ranges in 2006 and 2007. We used amplified fragment length polymorphisms (AFLPs) to assess which biotypes established in California, Oregon, and Montana at sites with varying climatic conditions, and whether the biotypes have hybridized in nature. Clustering and assignment tests showed that the Italian biotype successfully established at all study populations including those characterized by continental, winter-cold climates. We also found hybrids of the two parental biotypes, which in one study location constituted 42% of the population. Our data indicate that the Italian biotype is sufficiently plastic or able to adapt relatively quickly to a range of environments. Any benefits of introducing the Swiss biotype are at this point unclear.