The family Goodeidae comprises 38 viviparous fishes distributed throughout Mexico and 4 oviparous species that inhabit the southwestern United States. More than 22 of these species have been reported as endangered. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but is especially challenging for use with live-bearing fishes. Sperm of goodeids are usually packed in spermatozeugmata (bundles), which are difficult to dissociate without damaging the sperm. In this study we evaluated cryopreservation of spermatozeugmata. Xenotoca eiseni was used as a research model to develop a protocol, which was subsequently tested with two other goodeids, Goodea atripinnis and Ataeniobius toweri. Sperm quality was evaluated with an activating solution (NaCl-NaOH solution at 300 mOsmol/kg and pH 11.8) and counting of activatable bundles (AB) expressed as percentage AB (%AB) and motility duration of individual sperm at 200-x magnification. Post-thaw %AB was tested by using as cryprotectants methanol, dimethyl sulfoxide (DMSO), and glycerol at 5, 10, and 15%, equilibration exposure times of 10, 20, 40, and 60 min, and cooling rates of 5, 10, 20, 30, and 40 ºC/min. Sperm bundles maintained their packed form (Figure 1) after thawing (before activation). A combination of 20 min equilibration in 10% DMSO with cooling at 10 ºC/min yielded the highest post-thaw %AB (91 ± 3%) and duration (140 ± 18 sec). Spermatozeugmata concentrations of 4 × 10⁶/ml, 2 × 10⁶/ml, 4 × 10⁵/ml, and 4 × 10⁴/ml yielded no significant differences in post-thaw %AB or duration. Extender solutions at 300 mOsmol/kg had significantly higher post-thaw %AB and duration than did those at 200, 250, 350, or 400 mOsmol/kg. Of these extenders, Hanks' balanced salt solution (HBSS) had the highest %AB (71 ± 2%) and duration (177 ± 33 sec) at 72 h after thawing compared to phosphate-buffered saline (PBS) (28 ± 9%, 99 ± 19 sec) and NaCl (0%, 0s). A specific protocol (10% DMSO, 20-min equilibration, 10 ºC/min cooling, 2 × 10⁶/ml spermatozeugmata, and 300 mOsmol/kg HBSS) yielded post-thaw values of 96 ± 9 %AB with 814 ± 14 sec duration for Goodea atripinni, and 66 ± 2 %AB with 726 ± 25 sec duration for Ataeniobius toweri. This is the first study of cryopreservation of sperm within spermatozeugmata for viviparous fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers.