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Combretastatin A4-phosphate (CA4P) is a vascular disrupting agent that was recently described for the treatment of solid canine tumors. Conventional echocardiography and pulsed wave tissue Doppler imaging did not reveal cardiotoxicity in dogs, however, the gold standard for assessing myocardial damage in humans receiving cardiotoxic chemotherapeutics is two-dimensional speckle-tracking echocardiography. The current study evaluated the cardiotoxic effect of a single dose of CA4P in dogs using peak systolic strain measurements and the variability of these measurements. Echocardiographic examinations of seven healthy beagles and five canine cancer patients that received CA4P were retrospectively reviewed. Peak systolic regional longitudinal strain (LSt), peak systolic regional circumferential strain (CSt), and peak systolic regional radial strain (RSt) were measured before and 24 h after administration of CA4P. Peak systolic strain measurements were compared to serum cardiac troponin I (cTnI). To quantify intra- and inter-observer measurement variability, seven echocardiographic examinations were selected and each strain parameter was measured by three observers on three consecutive days. After CA4P administration, the median LSt and CSt values decreased by 21.8% (p = 0.0005) and 12.3% (p = 0.002), respectively, whereas the median RSt values were not significantly different (p = 0.70). The decrease in LSt was correlated with increased serum cTnI values (Spearman rho = −0.64, p = 0.02). The intra-observer coefficients of variation (CV) were 9%, 4%, and 13% for LSt, CSt, and RSt, respectively, while the corresponding interobserver CVs were 11%, 12%, and 20%. Our results suggest that regional peak systolic strain measurements may be useful for the early detection of cardiotoxicity that is caused by vascular disrupting agents and that LSt may be promising for the follow-up of canine cancer patients.