Objectives: The primary objective of this study was to compare laboratory practices for screening blood donors for syphilis at blood transfusion facilities in Ghana with the recommendations of the World Health Organization and the National Blood Service, Ghana (NBSG). The prevalence of syphilis antibodies in blood donors in Ghana was also estimated. Methods: Over an 11-month period, from February 2014 to January 2015, a semi-structured questionnaire was administered to 122 laboratory technical heads out of a total of 149 transfusion facilities in Ghana. The response rate was 81.9%. Results: A total of 58 (48%) transfusion facilities tested donors for syphilis, with an estimated 3.7% seroprevalence (95% confidence interval 3.6–3.8%). A total of 62 782 out of 91 386 (68.7%) donations were tested with assays that are not recommended. The estimated syphilis seroprevalence in voluntary donations was 2.9%, compared to 4.0% in family donations (p = 0.001). Only 6.9% of the health facilities were using standard operating procedures (SOPs). Conclusions: Despite international and national recommendations, more than half of the studied health facilities that provide blood transfusions in Ghana are not screening blood donations for syphilis. These data show a considerable mismatch between recommendations and practice, with serious consequences for blood safety and public health.