Triatoma infestans (Hemiptera: Reduviidae) from 22 Andean localities in Bolivia (n=968) and Peru (n=37) were analysed by multi‐locus enzyme electrophoresis. Among 12 gene–enzyme systems analysed, GPD, 6GPD and PGM were polymorphic, ACON, G6PD, GPI, 1DH, LAP, MDH, ME, PEP‐A and PEP‐B were monomorphic. Allozyme frequencies were analysed in relation to geographical and climatic factors, and the presence or absence of Trypanosoma cruzi infection. At one locality (Vallegrande, Bolivia), the frequency of 6Pgd‐1 was significantly higher in infected (41% of 85) than in uninfected (17% of 83) adult T. infestans, although no such difference was found among nymphs (n = 347). From other localities, only insects infected with T. cruzi were subjected to isozyme analysis. Populations of T. infestans within villages showed panmixia, while genetic differentiation of T. infestans between villages was correlated with the distance between them. The genetic structure of T. infestans natural populations followed an ‘isolation by distance’ model, involving a series of founder effects followed by genetic drift, rather than adaptation in response to differential selection pressures. This conforms with circumstantial evidence that T. infestans spread, mainly in association with recent human migrations, from a source, probably in southern Bolivia. Isoenzyme characterization of populations of T. infestans could be used to infer sources of re‐infestation during the surveillance phase of control programs.