Background: The aim of the study was to investigate the value of longitudinal follow-up of dobutamine thallium-201 single photon emission tomography (<sup>201</sup>Tl SPECT) in the development of significant cardiac allograft vasculopathy (CAV) after orthotopic heart transplantation. Methods: We studied 38 cardiac recipients (mean age 57 ± 12 years) who underwent at least two follow-up dobutamine <sup>201</sup>Tl SPECT since January 1998. All patients had normal coronary angiography and normal left ventricular function initially. Results: After 2.3 ± 1.8 years, 12 patients developed significant CAV and there were 4 cardiac deaths (1 died suddenly). Of the 99 scans retrospectively analyzed, patients with significant CAV had elevated values of inhomogeneity score, lung/heart ratio (LHR) at stress and lower left ventricular ejection fraction (all p < 0.05). The higher values of inhomogeneity were significantly correlated with higher stress LHR (r = 0.301, p = 0.021), and lower ejection fraction (r = –0.379, p < 0.001). Progressive inhomogeneity was noted in all heart recipients, and more rapid, although statistically insignificant, in patients who developed significant CAV. Ten patients had inadequate chronotropic response to dobutamine infusion up to 40 µg/kg/min in the follow-up studies. The late onset of chronotropic incompetence was an independent predictor of CAV development (p = 0.03). Conclusions: Progressive inhomogeneity of myocardial perfusion, higher lung uptake at stress and chronotropic incompetence assessed by dobutamine <sup>201</sup>Tl myocardial SPECT provide incremental diagnostic value in detecting significant CAV.