Background: Botswana has a shortage of health care workers, especially in primary healthcare. Retention and high performance of employees are closely linked to job satisfaction and motivation, which are both highest where employees’ personal values and goals are realised. Aim: The aim of the study was to evaluate employees’ personal values, and the current and desired organisational culture of the district health services as experienced by the primary health care workers. Setting: The study was conducted in the Ngamiland and Mahalapye health districts. Method: This was a cross sectional survey. The participants were asked to select 10 values that best described their personal, current organisational and desired organisational values from a predetermined list. Results: Sixty and 67 health care workers completed the survey in Mahalapye and Ngamiland districts, respectively. The top 10 prevalent organisational values experienced in both districts were: teamwork, patient satisfaction, blame, confusion, job insecurity, not sharing information and manipulation. When all the current values were assessed, 32% (Mahalapye) and 36% (Ngamiland) selected by health care workers were potentially limiting organisational effectiveness. The organisational values desired by health care workers in both districts were: transparency, professional growth, staff recognition, shared decision-making, accountability, productivity, leadership development and teamwork. Conclusions: The experience of the primary health care workers in the two health districts were overwhelmingly negative, which is likely to contribute to low levels of motivation, job satisfaction, productivity and high attrition rates. There is therefore urgent need for organisational transformation with a focus on staff experience and leadership development.