Up-flow biological activated carbon (BAC) filters have been empirically employed in drinking water treatment plants (DWTPs) to address the challenges of its down-flow counterparts (e.g., high head loss and insufficient use of BAC beds), yet their performances and mechanisms toward ammonia removal are not fully evaluated. This study characterized the occurrence, distribution, and diversities of nitrifiers in up-flow and down-flow BAC filters by investigating 18 full-scale drinking water treatment trains in different geographic locations. Quantitative polymerase chain reaction analysis of gene markers of target microorganisms demonstrated higher numbers of total bacteria, ammonia-oxidizing bacteria (AOB), and Nitrospira in the up-flow filters relative to the down-flow filters (P < 0.05), implying enhanced biological activities and nitrification potential within up-flow filters. The dominance of ammonia-oxidizing archaea (AOA) over AOB (i.e., 1.3-4.0 log10 gene copies higher) in 17 BAC filters illustrated the critical role of AOA in drinking water nitrification. Stratification of biomass was mainly found in the down-flow filters rather than the up-flow filters, suggesting better mixing of filter media across up-flow filter beds. Analysis of similarity results revealed that the AOA and Nitrospira community compositions were mainly affected by water sources and locations (P < 0.05) but not flow configurations. These results provide insight into nitrification mechanisms in BAC filters with different flow configurations in real-world DWTPs.