Objective HIV-infected women on antiretroviral therapy have a higher risk of preterm birth than HIVuninfected women in Botswana. To better understand the mechanism for preterm birth among HIV-infected women, we evaluated whether mid-trimester cervical length differed by HIV status as cervical shortening is associated with an increased risk for preterm birth. Methods We conducted a prospective cohort study among pregnant women receiving care at the Scottish Livingstone Hospital in Molepolole, Botswana. Consecutive women referred for routine obstetrical ultrasound were consented and enrolled if between 22w0d and 24w6d by ultrasound biometry. Blinded to maternal HIV status, an obstetrician measured transvaginal cervical length using standardized criteria. Cervical length, as well as the proportion of women with a short cervix (<25mm), were compared among HIV-infected and HIV-uninfected women. The acceptability of transvaginal ultrasound was also evaluated. Results Between April 2016 and April 2017, 853 women presenting for obstetric ultrasound were screened, 187 (22%) met eligibility criteria, and 179 (96%) were enrolled. Of those enrolled, 50 (28%) were HIV-infected (86% on antiretroviral therapy), 127 (71%) were HIV-uninfected, and 2 (1%) had unknown HIV status. There was no significant difference in mean cervical length between HIV-infected and HIV-uninfected women (32mm vs 31mm, p =