Fragipans are subsurface soil layers conducive to denitrification at a depth where nitrate has likely reached beyond the plant root zone. Previous research indicates that surface manure addition could increase denitrifiers at all soil depths and cause stratification above fragipans. Techniques based on functional genes involved in denitrification have been used to study the highly diverse denitrifying bacteria in various environments. In our study, denitrifier community was evaluated in fragipan soils from a formerly cultivated soil with poultry litter amendment and an unamended pasture site. DNA was extracted from intact soil cores from the surface to the depth of the fragipan. Three denitrifier-specific primers were used for the PCR amplification: nirS and nirK, encoding nitrite reductase and nosZ, encoding nitrous oxide reductase. Skim milk greatly improved DNA recovery by commercial kits for both surface and subsurface soil samples. DNA concentrations measured by nanodrop technique were significantly different between the surface and subsurface soils, ranging from 3 µg/ml to 128 µg/ml, and were stratified in some samples. The amplification of extracted DNA using nirS and nirK primers was successful in some soil samples, but not successful or non specific in other samples. The nosZ primer we used resulted in non specific bands for most samples. The failed amplification may be because of low DNA concentration or the potential for unique denitrifier community composition, which was investigated by DGGE. Our study suggests that population and potential community composition differences of denitrifiers occur in fragipan soils relative to surface soils.