Studies of in vitro fertilization (IVF) and sperm cryopreservation have been conducted in several small cat species, but virtually no data exist for black-footed cats (Felis nigripes) (BFCs) or sand cats (Felis margarita) (SCs). The objectives of this study were 1) to compare in vitro motility and acrosome status of fresh and cryopreserved (frozen in pellets on dry ice or in straws in liquid nitrogen vapor) BFC and SC spermatozoa cultured in feline-optimized culture medium (FOCM) or Ham F-10, 2) to assess ovarian responsiveness in BFCs and SCs following exogenous gonadotropin treatment and laparoscopic oocyte recovery, and 3) to evaluate the fertility of fresh and frozen-thawed spermatozoa from both species using homologous and heterologous (domestic cat oocytes) IVF in the two culture media. Motility and acrosomal integrity of fresh and frozen-thawed spermatozoa from BFCs and SCs were similar (P > 0.05) in both media during 6 h of culture. Although effects were more pronounced in SCs, cryopreservation in straws was superior (P < 0.05) to cryopreservation in pellets for both species. Gonadotropin stimulation produced ∼16 ovarian follicles per female, and >80% of recovered oocytes were of optimal (grade 1) quality. The BFC and SC spermatozoa fertilized 60.0%–79.4% of homologous and 37.7%–42.7% of heterologous oocytes in both culture media, with increased (P < 0.05) cleavage of homologous (SC) and heterologous (BFC and SC) oocytes in FOCM. These results provide the first information to date on the gamete biology of two imperiled cat species and further our capacity to apply reproductive technologies for their conservation.