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Ambystoma mexicanum is in danger of extinction in free-living, due to anthropogenic actions; sperm cryopreservation for captive breeding can help in its ex-situ conservation. This research aimed to identify the viability of fresh and post-thawing sperm from different spermatophores. During the breeding season, spermatophores releasing was induced in nine specimens by reducing water temperature. The mean concentration per spermatophores was 2.6 ± 0.6 x104 sperm. A reduction of 30 % of living sperms and an increase of 15 % of abnormal morphology were determined in fresh and post-thawing sperm. With the WGA and PNA lectins bounded to FITC, two different fluorescence patterns were determined in each one, which showed the membrane presence and distribution of N-acetyl glucosamine, sialic acid, and mannose respectively. Sperm percentages in each fluorescence pattern showed differences associated with the number of spermatophores in each release. Differences in sperm viability from releases with different numbers of spermatophores were determined. The sperm collection and cryopreservation protocol of A mexicanum were efficient as tools for using cryopreserved semen for ex situ reproduction. [ABSTRACT FROM AUTHOR]