Molecular based approaches to assess microbial biomass and diversity from soil and other ecosystems are rapidly becoming the standard methodology for analysis.  While these techniques are advantageous because they do not rely on the need to culture organisms, each technique may have its own biases and/or limitations that need to be addressed prior to full-scale implementation.  We have chosen a PCR based approach (qPCR-LH) that consists of two steps: an initial quantitative PCR (qPCR) followed by length heterogeneity analysis (LH) using a fluorescence-based automated capillary DNA sequencer.  To date we have focused our analysis on fungi using a variety of conserved rRNA primers.  DNA extracts obtained from pure cultures of soil fungi (> 25 species) analyzed singly and in mixtures have been used to address some of the methodology considerations: primer specificity, PCR efficiency, and length heterogeneity.  In addition, community profiles from a variety of field studies will also be discussed.