Historically, semen banking in felids has required sample collection via electroejaculation followed by sperm freezing in straws over LN₂ vapor. Recent modifications include urethral catheterization of males treated with α-2 agonists for semen recovery and vitrification of cat sperm by suspension in a sucrose-based cryomedium and direct pelleting into LN₂. In combination, these latter methods greatly simplify semen cryopreservation in cats but protocols need to be optimized for applied usage. In the present study, our goal was to assess the effect of 2 variables—pellet volume and thawing temperature—on post-thaw sperm motility, acrosome status, and in vitro fertility. Semen was collected from 3 males (3 ejaculates/male) via urethral catheterization under dexmedetomidine-ketamine anaesthesia. Sperm were diluted in Feline Optimized Culture Medium (FOCM), centrifuged (8 min; 300 × g), and resuspended in a soy-lecithin-based vitrification medium (with 0.2 m sucrose). After a 5-min equilibration, sperm was vitrified in 2 volumes (20 or 30 µL) by direct pipetting into LN₂. Sperm pellets were thawed in FOCM at 1 of 2 temperatures (37 or 55°C) and the 4 treatment groups (20 µL-37°C, 20–55, 30–37, 30–55) assessed for percentage of progressively motile and acrosome intact sperm. To assess sperm function, additional 30-µL pellets were thawed at 37 or 55°C, and recovered sperm were used to inseminate in vitro-matured domestic cat oocytes (n = 10–25/ejaculate). At 48 h post-insemination, oocytes and embryos were fixed (1% NBF). Hoechst fluorescent stain (#33342) was used to evaluate embryo cleavage and maturation status of unfertilized ova. Sperm motility and acrosomal integrity percentages were analysed by ANOVA, and oocyte cleavage proportions were analysed by chi-squared. Mean (± SEM) progressive sperm motility post-thaw did not differ (P > 0.05) among treatments (38 ± 8, 34 ± 7, 41 ± 7, 32 ± 7% for 20 µL-37°C; 20–55, 30–37, and 30–55, respectively). Similarly, acrosomal integrity did not differ (P > 0.05) among treatments (26 ± 4, 25 ± 4, 17 ± 3, 17 ± 2% for 20 µL-37°C, 20–55, 30–37, and 30–55, respectively). Oocyte cleavage proportions did not differ (P > 0.05) between thawing temperatures for total inseminated oocytes but, after correcting for oocyte maturation status, was higher (P < 0.01) for samples thawed at 55°C (60%, 67/112) compared with 37°C (39%, 52/133). In summary, although variations in pellet volume and thawing temperature had minimal effect on sperm motility or acrosome status immediately post-thaw, sperm function appeared to be enhanced when vitrified pellets were thawed at a higher temperature. In vitro fertility success (~60% embryo cleavage) is comparable to values reported by our laboratory with conventionally collected and frozen cat semen, suggesting these newer methods may be suitable for applied usage in felids.