Urethral catheterization (Zambelli method) is an alternative technique used for semen collection through electroejaculation. Results recorded for wild felids have shown its efficacy in ten felid species, despite the considerable variation between individuals and small number of samples in some studies. Given the innovative potential of the urethral catheterization technique, the aim of the present study was to evaluate this semen collection protocol followed by electroejaculation, applied to jaguarundi (Puma yagouaroundi) and to compare it to electroejaculation, alone. Semen collections (n = 5) were carried out in males (n = 7). Semen was collected through urethral catheterization (UC), which was followed by electroejaculation (UC-EEJ, n = 3), or alternatively by electroejaculation (EEJ, n = 2) collections. The UC technique was effective in semen collection in 57 % of animals (n = 4, but none of the animals was able to provide semen in all collections. Results are expressed as mean± SD. Six semen samples were collected by UC within 18 collection trials-mean volume of 3.53 ± 0.69 µL was reached, but only one sample showed the necessary quality for the freezing procedure (vigor 3, 80 % motility and 88 % vitality). The other samples showed 1.86 ± 0.86 vigor, 21.32 ± 12.59 % motility and 27.6 ± 13.05 % vitality. It was not possible assessing samples’ semen concentrations due to their low volume. UC-EEJ and EEJ samples recorded vigor 3 ± 0.93 and 3.13 ± 0.67; motility 52.50 ± 25.36 % and 54.09 ± 20.47 %; and vitality 58.50 ± 24.50 % and 62.78 ± 16.28 %, respectively. These samples accounted for better qualitative parameters than UC samples, but they did not differ from each other. In conclusion, the UC technique was effective in semen collection, but the quality of samples collected through it did not meet the standards for cryopreservation; their values were lower than those of the two tested electroejaculation modalities.