Abstract Background Injecting drug users (IDU) remain an important population at risk for blood-borne infections such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). In the Netherlands, a program is being implemented to offer annual voluntary screening for these infections to opioid drug users (ODUs) screened in methadone care. At two care sites where the program is now operating, our study aimed to estimate the seroprevalence among ODUs screened for HIV, HBV and HCV; to evaluate HBV vaccination coverage; and to assess the feasibility of monitoring seroprevalence trends by using routine annual screening data. Methods Opioid drug users on methadone treatment are routinely offered voluntary screening for infectious diseases such as HIV, HBV and HCV. Data on uptake and outcome of anti-HIV, anti-HBc, and anti-HCV screening among ODUs receiving methadone were obtained from two regions: Amsterdam from 2004 to 2008 and Heerlen from 2003 to 2009. Findings Annual screening uptake for HIV, HBV and HCV varied from 34 to 69%, depending on disease and screening site. Of users screened, 2.5% were HIV-positive in Amsterdam and 11% in Heerlen; 26% were HCV-positive in Amsterdam and 61% in Heerlen. Of those screened for HBV, evidence of current or previous infection (anti-HBc) was found among 33% in Amsterdam and 48% in Heerlen. In Amsterdam, 92% were fully vaccinated for HBV versus 45% in Heerlen. Conclusion Annual screening for infectious diseases in all ODUs in methadone care is not fully implemented in the Netherlands. On average, more than half of the ODUs in methadone care in Heerlen and Amsterdam were screened for HIV, HBV and HCV. In addition, screening data indicate that HBV vaccination uptake was rather high. While the HIV prevalence among these ODUs was relatively low compared to other drug-using populations, the high HCV prevalence among this group underscores the need to expand annual screening and interventions to monitor HIV, HBV and HCV in the opioid drug-using population.