Microbial community structure was assessed in a horizontal subsurface flow planted sand filter treating domestic wastewater with molecular and culture-based methods. The diversity and spatial distribution of the microbial community was investigated using a PCR-DGGE (eubacterial and archaeal primers, ammonia-oxidizing bacteria, and ammonium monooxygenase specific primers), and spread plate and MPN counts. Significant differences were found in the spatial distribution of the microbial community structure. Data analysis revealed that different components of the microbial community possessed different spatial distribution patterns within the filter bed and depending on community type, relationships with soil chemical, and microbiological parameters varied. The most important spatial pattern in microbial community structure within the constructed wetland was related to the depth gradient, followed by differences between inflow and outflow. A comparison of a number of heterotrophic bacteria between inlet and outlet pipes as well as between two sampling depths showed no significant differences. In addition, the variation of the abundance of ammonia-oxidizing bacteria demonstrated no clear spatial pattern.