Exercise therapy at the aerobic level is highly recommended to improve clinical outcomes in patients with heart failure, in which cardiopulmonary exercise testing (CPX) is required to determine anaerobic thresholds (ATs) but is not available everywhere. This study aimed to validate a method to estimate the AT using heart rate variability (HRV) analysis from electrocardiography data in patients with heart failure. Between 2014 and 2019, 67 patients with symptomatic heart failure underwent CPXs in a single university hospital. During the CPX, RR intervals was measured continuously and the HRV threshold (HRVT), defined as the inflection point of <5 ms(2) of a high-frequency component (HFC) using the power spectrum analysis, was determined. Patients were divided into two groups according to the mean HFC at rest (high-HFC group, n = 34 and low-HFC group, n = 33). The high-HFC group showed good correlation between the VO(2) at AT and HRVT (r = 0.63, p < 0.001) and strong agreement (mean difference, -0.38 mL/kg, p = 0.571). The low-HFC group also showed modest correlation (r = 0.41, p = 0.017) but poor agreement (mean differences, 3.75 mL/kg, p < 0.001). In conclusion, the HRVT obtained from electrocardiography may be a useful indicator for estimating AT in patients with heart failure.