Glomalin was measured in soil from farming systems managed for 8 years by chisel tillage (CT), more intensive tillage for organic (ORG) production, and no tillage (NT) on Acrisols (FAO Soil Units) in the Mid-Atlantic region of the U.S. Whole soil and aggregate size classes of >2.00, 0.50–2.00 and 0.21–0.50 mm (macroaggregates), 0.05–0.21 mm (microaggregates), and <0.05 mm (fine material) were examined. Glomalin-related soil protein (GRSP) was extracted from 1-g samples (four plots per treatment) with 100 mM sodium pyrophosphate, pH 9.0, at 121 °C in three extraction cycles. Extracts were pooled and quantified by using the Bradford protein assay. Concentrations of GRSP and total carbon (C) in aggregates were linearly related across aggregate size classes for all treatments (GRSP = 0.101C + 0.56, r2 = 0.95). No tillage had significantly greater whole soil GRSP than did CT or ORG (P = 0.01). Mean values for GRSP in aggregates of NT were higher than for CT or ORG aggregates by 0.53 and 0.66 mg g−1 aggregates, respectively. There were no differences among treatments in GRSP concentrations in fine material. In NT the concentration of GRSP increased as aggregate size increased in contrast to the disturbed treatments, CT or ORG, where there were no differences in GRSP concentration across aggregate size fractions. Larger proportions of GRSP were distributed in macroaggregates of NT compared to CT and ORG in contrast to larger proportions in microaggregates of CT and ORG than in NT. Although soil disturbance in ORG farming is greater than for CT farming, both treatments had similar GRSP concentrations and distributions.