<b>Background:</b> South Africa is experiencing the worst HIV-driven tuberculosis (TB) epidemic in the world. More than 300,000 new cases of active TB are reported in the country each year with 60% co-infected with HIV. Isoniazid preventive therapy (IPT) is a key public health intervention for the prevention of TB among people living with HIV (PLHIV) and is recommended as part of a comprehensive HIV and AIDS care strategy. However, program data suggests that coverage of IPT service to be very low. This study aims to assess IPT initiation rate among newly diagnosed HIV-positive persons in three high HIV-burden districts of South Africa. <b>Methods: </b>A cross-sectional study was conducted using routine data generated from pre-ART and ART programs in 35 purposively selected primary health care (PHC) clinics in South Africa. The facilities were selected from three high HIV-burden districts with a mix of urban and rural settings. TB screening and IPT initiation status was assessed within a window period of one-year post HIV diagnosis. Initiation rate of IPT services among newly diagnosed HIV-positive persons was assessed. The chi-squared test was used to determine whether there was a significant difference in the proportion of newly diagnosed HIV-positive persons who were initiated on IPT by sex, age group, pregnancy status, health facility, district and location of facility. <b>Results:</b> We identified 12,413 newly diagnosed HIV patients aged 12-years-old and above between June 1, 2014 and March 31, 2015. TB screening was not conducted among 33% of newly diagnosed HIV-positive persons to rule out or confirm the presence of active TB. IPT was initiated in 42.2% of known IPT-eligible HIV-positive persons. Initiation of IPT services was lower in younger patients aged 12 to 20-years-old compared to older patients. The proportion of pregnant women who were initiated on IPT was higher compared to the proportion in non-pregnant women (51.0% and 40.1% respectively;P < 0.05). Health care clinics located in uThukela health