Although studies have demonstrated reasonable pregnancy rates in single-ovulating heifers inseminated with sexed sperm, it is not yet clear if sexed sperm can be successfully used in superovulated females. Therefore, the objective of this trial was to determine fertilization rate and embryo quality in superovulated heifers inseminated with frozen-thawed Xchromosome bearing sperm at 2 different dosages versus unsorted sperm. Nulliparous Holstein heifers (n = 23; 12 to 16 mo) were superovulated with FSH and observed for estrus twice a day. Following detection of estrus, bilateral deep uterine horn AI was performed once (1X) or twice (2X) in each heifer, according to the treatments: S20-1X: AI with 20 million X-sorted sperm 12 h after the onset of estrus; S10-2X: AI with 10 million X-sorted sperm 12 and 24 h after the onset of estrus; and U10-2X: AI with 10 million unsorted sperm 12 and 24 h after the onset of estrus. Embryos/ova were collected 7 d after estrus and evaluated. Ten d later, heifers were resynchronized for a second superovulatory treatment and assigned to a second treatment group. Data were compared using mixed effects models. When sexed sperm were used, number and percentage of fertilized and viable embryos recovered per flush was similar between the S20-1X and S10-2X group (63.5% and 61.9%), but lower (P < 0.01) than in the U10-2X group (90.9%). In addition, heifers bred with X-sorted sperm had an increase (P < 0.05) in the percentage of degenerate embryos when only fertilized structures were included in the analysis (S20-1X = 58.6%, S10-2X = 53.1%, U10-2X = 24.2%). In conclusion, it seems likely that the sorting process may have caused damage in the sperm that compromised fertilization as well as subsequent embryonic development in superovulated heifers.