Technical Abstract: Agricultural drainage ditches have large differences in hydroperiod and provide additional water residence time to mitigate farm runoff before it reaches receiving water bodies. These ditch wetland habitats harbor a characteristic benthic macroinvertebrate fauna reflective of the assimilative capacity and sometimes brief ditch hydroperiod. Ten agricultural ditch sites were characterized for water and sediment quality, macroinvertebrate community composition, and other habitat parameters throughout a 100 m reach on each ditch. Seasonal differences in water quality between different ditch size classifications existed. Macroinvertebrate collections yielded 68 different taxa, and seasonal differences were also noted among ditch sizes. Mean annual Percent Chironomidae / Total Diptera scores were similar in most ditch sizes ranging from 85 ± 4% in quaternary ditches to 98 ± 1% in secondary ditches. Using Jaccard’s Index of Similarity, tertiary and riverine ditches were most similar (64%), while primary and tertiary ditches were least similar (31%). A cluster analysis was performed comparing the Hilsenhoff Biotic Index (HBI), taxa richness, Shannon-Wiener, and percent Ephemeropta, Plecoptera, Tricoptera (EPT) metrics. When combined with physical environmental quality measurements, benthic macroinvertebrates may be good indicators of water body changes. The environmental value of certain best management practices can be validated and related to resident organisms that reflect the range of conditions specific to each drainage. However, baseline information on resident communities of unique ecosystems, such as agricultural drainage ditches, must first be established.