The natural hierarchy of rivers contains microhabitat, habitat, stream reach, stream segment, river sub-basin (stream basin), and river basin scales. The  aim  of  our study was to assess the stream benthic macroinvertebrate  assemblage patterns among headwater segments within a  tropical  watershed.  The  sampling  was  performed  during  the  dry season/2006,  in  three  streams located at National  Park  of  Serra  do  Cipó,  Brazil. Two reaches of each stream were sampled.  In each reach, three riffles (habitats);   and   in   each   riffle,   three   types   of  substrates. A total of 64,793 individuals were collected and identified (96% insects).  The ANOSIM showed the greatest differences   in  assemblage structure  to  be  associated  with  stream segment and  substrate types. These results were confirmed by cluster analysis, NMS ordination and indicator species analysis. The diversity additive partitioning analysis revealed that there  was  high beta diversity in taxa richness among stream segments, much  greater than expected by random. We  conclude that  accurate regional biological assessment of benthic diversity in tropical headwater streams can to  some  extent  ignore  reach and riffle location  within each headwater stream, concentrating  sampling  effort on obtaining samples from many headwater streams,  and  from  the  variety  of  substrate types found within each stream.