Heart rate variability (HRV) is a well established mortality risk factor in both healthy dogs and those with heart failure. While the standards for short-term HRV analysis have been developed in humans, only reference values for HRV parameters determined from 24-hour ECG have been proposed in dogs. The aim of this study was to develop the reference values for short-term HRV parameters in a group of 50 healthy dogs of various breeds (age 4.86 ± 2.74 years, body weight 12.2 ± 3.88 kg). The ECG was recorded continuously for at least 180 min in a dark and quiet room. All electrocardiograms were inspected automatically and manually to eliminate atrial or ventricular premature complexes. Signals were transformed into a spectrum using the fast Fourier transform. The HRV parameters were measured at fixed times from 60-min ECG segments. The following time-domain parameters (ms) were analyzed: mean NN, SDNN, SDANN, SDNN index, rMSSD and pNN50. Moreover, frequency-domain parameters (Hz) were determined, including very low frequency (VLF), low frequency (LF) and high frequency (HF) components, total power (TP) and the LF/HF ratio. The results (means ± SD) were as follows: mean NN = 677.68 ± 126.89; SDNN = 208.86 ± 77.1; SDANN = 70.75 ± 30.9; SDNN index = 190.75 ± 76.12; rMSSD = 259 ± 120.17, pNN50 = 71.84 ± 13.96; VLF = 984.96 ± 327.7; LF = 1501.24 ± 736.32; HF = 5845.45 ± 2914.20; TP = 11065.31 ± 3866.87; LF/HF = 0.28 ± 0.11.