BACKGROUND: In malaria endemic areas, individuals are frequently asymptomatic and may be undetected by conventional microscopy or newer, rapid diagnostic tests. Molecular techniques allow a more accurate assessment of this asymptomatic parasite burden, the extent of which is important for malaria control. This study examines the relative prevalence of sub-microscopic level parasite carriage and clonal complexity of infections (multiplicity of infection) over a range of endemicities in a region of north-eastern Tanzania where altitude is an established proxy of malaria transmission. The PCR prevalence was then compared against other measures of transmission intensity collected in the same area. METHODS: This study used 1,121 blood samples collected from a previously conducted cross-sectional malario-metric survey during the short rainy season in 2001 from 13 villages (three at < 600 m, four at 600-1,200 m and six at > 1,200 m in altitude above sea level). Samples were analysed by PCR for carriage of parasites and multiplicity of infection. These data were compared with other measures of transmission intensity collected from the same area. RESULTS: Parasite prevalence was 34.7% by PCR and 13.6% by microscopy; a 2.5-fold difference in line with other recent observations. This fold difference was relatively consistent at the different altitude bands despite a marked decrease in parasite prevalence with altitude: < 600 m 70.9 vs 28.6, 600-1,200 m 35.5 vs 9.9, > 1,200 m 15.8 vs 5.9. The difference between parasite prevalence by PCR was 3.2 in individuals aged between 15 and 45 years (34.5 vs 10.9) compared with 2.5 in those aged 1-5 (34.0 vs 13.5) though this was not statistically significant. Multiplicity of infection (MOI) ranged from 1.2 to 3.7 and was positively associated with parasite prevalence assessed by both PCR and microscopy. There was no association of MOI and age.Village level PCR parasite prevalence was strongly correlated with altitude, sero-conversion rate and predicted entomological inoculation rate. CONCLUSIONS: Asymptomatic, low density, multi-clone malaria infection was common in this study area. These infections are important as potential contributors to the infectious reservoir of parasites and need to be identified by control programmes especially in this era where malaria elimination is a focus. High throughput standardized PCR approaches are needed to identify individuals who are malaria carriers.