Women living with HIV are faced with lack of adequate information about HIV prevention. This study assessed the level of knowledge of prevention of mother-to-child transmission (PMTCT) in Serowe, Botswana. The objectives of this study were to assess the level of knowledge of the PMTCT programme and vertical transmission among HIV-positive women in Serowe, Botswana and as well as describe the practices of family planning among the participants. The study was a cross-sectional descriptive survey using qualitative method. Twenty six (26) participants attending PMTCT clinics at Serowe clinic, Nutrition clinic, and Kadimo clinic participated in the study between October and December 2008 after consenting to participate. In-depth interviews were conducted to collect socio-demographic data of the participants. Qualitative methods were used to collect data about the women’s level of understanding of the PMTCT programme and family planning practices. The interviews were conducted in the local language (Setswana) and translated into English by bilingual experts and participants’ responses were audio taped. Although all 26 participants had good knowledge and understanding of the family planning practices, but only 18 (69.2%) had used the family planning methods. The findings in this study revealed good social support from the family members and the disclosure of HIV status was not a big issue to the participants. Majority of respondents (66.6%) had good knowledge and understanding of the PMTCT programme. One-third of the participants were single, 96% of participants were unemployed and about one-third of them live on less than 50 U$ dollars per month. The use of family planning by participants was poor despite their good knowledge about prevention of mother-to-child transmission.