The gonads of six adult giant otter males (Pteronura brasiliensis) and of one adult neotropical otter male (Lontra longicaudis) were analyzed both for histology and gross anatomy. The mean testes mass/body mass ratio in giant otters was 0.046 ± 0.0071%. The presence of spermatozoa inside the seminiferous and epididymal tubules revealed that two-year-old giant otters were already sexually mature. The mean diameter of the seminiferous and epididymal tubules of mature giant otters was 126.3 ± 13.37 μm and 198.8 ± 31.19 μm, respectively. The small amount of spermatozoa in the testes and epidimydes of 5 out of 6 giant otters analyzed suggests a seasonal testes activity in this species, which is in accordance to the observations of a monogamous mating system and seasonal reproduction of free-ranging giant otters. The testes of the neotropical otter were proportionally larger, wider and heavier than the testes of the giant otters. The testes mass/body mass ratio in L. longicaudis was 0.25%, 5.5 times greater than that of giant otters. The mean diameter of the seminiferous and epididymal tubules of the neotropical otter were 179.8 ± 17.01 μm and 238.5 ± 24.64 μm, respectively, which were significantly larger than those of the giant otter. The differences in testes size between these two otter species might be related to different mating systems. The greater dimensions of the testes of the neotropical otter, together with the greater body size of males when compared to females, suggest a non-monogamous mating system in this species, probably polyginy or promiscuity, which, however, needs to be better investigated.