Maternal tobacco smoking is a recognized risk behavior that has adverse impacts on maternal and fetal health. However, in some populations, the use of smokeless tobacco exceeds the use of smoked tobacco. In central Australia, Aboriginal populations utilize wild tobacco plants (Nicotiana spp.) as a smokeless product. These plants are known by a variety of names, one of which is pituri. The plants are masticated and retained in the oral cavity for extended periods of time and their use continues throughout pregnancy, birth, and lactation. In contrast to the evidence related to combusted tobacco use, there is no evidence as to the effects of pituri use in pregnancy. Central Australian Aboriginal women who were at least 28 weeks pregnant were stratified into three tobacco exposure groups: (a) Pituri chewers, (b) smokers, and (c) non-tobacco users. Routine antenatal and birth information, pre-existing and pregnancy-related maternal characteristics, fetal characteristics, and biological samples were collected and compared. The biological samples were analysed for tobacco and nicotine metabolite concentrations. Samples from the mother included venous blood, urine, hair and colostrum and/or breast milk. From the neonate, this included Day 1 and Day 3 urine and meconium, and from the placenta, arterial and venous cord blood following delivery. This is the first study to correlate the pregnancy outcomes of central Australian Aboriginal women with different tobacco exposures. The findings will provide the foundation for epidemiological data collection in related studies. Note to readers: In this article, the term “Aboriginal” was chosen by central Australian women to refer to both themselves and the Aboriginal people in their communities. “Indigenous” was chosen to refer to the wider Australian Aboriginal and Torres Strait Islander people.