Approximately 25% of dairy heifers subjected to timed AI are observed in estrus 24 h before prescheduled AI, creating an asynchrony between ovulation and insemination. Objectives were to evaluate the effects of timing of AI and use of 2 semen extenders (SE) on pregnancy per AI (P/AI) and pregnancy loss. Holstein heifers (≥13 mo of age, body weight ≥350 kg, wither height ≥122 cm) received GnRH and an intravaginal insert containing progesterone on study d −8. Inserts were removed 5 d later and heifers received injections of PGF_2α on d −3 and −2, followed by GnRH on d 0. Heifers were assigned randomly to receive timed AI with untreated semen on d 0 (72 h after first PGF_2α; positive control; 72-CON; n = 103), timed AI with untreated semen on d −1 (48 h after first PGF_2α; negative control; 48-CON; n = 100), timed AI on d −1 with SE1-treated semen (48-SE1; n = 98), timed AI on d −1 with SE2-treated semen (48-SE2; n = 102). A total of 4 bulls were used. Sample size was sufficient to detect a 20-percentage point difference among treatments (55 vs. 35%; α = 0.05; β = 0.20). Heifers were fitted with automated estrus detection monitors. Pregnancy was diagnosed by transrectal ultrasonography 29 and 54 d after AI. Data were analyzed by logistic regression. Statistical models included fixed effects of treatment and enrollment week. Orthogonal contrasts were used to assess the effects of day of AI (72-CON vs. 48-CON+48-SE1+48-SE2), use of semen extenders (48-CON vs. 48-SE1+48-SE2), and extender type (48-SE1 vs. 48-SE2). Onset of estrus between 24 and 48 h after first PGF_2α was detected in 66.6% of heifers. Pregnancy per AI on d 29 (72-CON = 60.1, 48-CON = 35.4, 48-SE1 = 39.8, 48-SE2 = 32.2%) and 54 (72-CON = 57.1, 48-CON = 31.7, 48-SE1 = 36.0, 48-SE2 = 29.7%) was greater (P < 0.001) for heifers inseminated on d 0 compared with d −1; however, no effect semen extender or extender type was observed. Treatment did not affect pregnancy loss (72-CON = 3.1, 48-CON = 7.9, 48-SE1 = 7.3, 48-SE2 = 5.6%). Hastening AI by 24 h decreased likelihood of pregnancy, which was not improved by the use of semen extenders.