Dalmanelloid brachiopod shells were collected from the Upper Ordovician Lexington Formation (lower Katian) of Kentucky, Sheguindah Shale (middle Katian) on Manitoulin Island, Ontario, and the Stony Mountain Formation (upper Katian) in the Winnipeg area, Manitoba. They were investigated to test the hypothesis of paleo-latitudinal zonation of the shelly benthos. A multi-technique approach was applied to evaluate the petrographic and geochemical (isotopic and elemental) preservation of the secondary layer of shells. Preliminary conventional microscopy, cathodoluminescence (CL), and scanning electron microscopy (SEM) confirmed the retention of primary shell ultrastructure (prismatic low-Mg calcite). The geochemical diagenesis proxies (e.g., Sr, Mn, Fe, and ΣREE) show insignificant correlations with the δ18O and δ13C values, thus supporting the preservation of at least near-primary geochemical compositions. Among the three lots of shells, the mean δ18O value is the highest in those from the Lexington Formation (–4.5‰ ± 0.3‰ VPDB), lowest from the Stony Mountain Formation (–6.8‰ ± 0.4‰ VPDB), and intermediate from the Sheguindah Shale (–6.0‰ ± 0.8‰ VPDB). The relative gradient in δ18O increase is in agreement with the paleo-latitudinal gradient, with Kentucky in subtropical, southern Ontario in mid-tropical, and southern Manitoba in subequatorial latitudes. The Lexington Formation shells also have the highest mean δ13C value (0.8‰ ± 0.2‰ VPDB) and relatively high P contents (170 ± 27 ppm), suggesting higher organic productivity, which is consistent with previous interpretation of frequent upwelling of nutrient-rich cool waters along the southeastern margin of Laurentia during the Katian. The Lexington shells also have a lower mean Th/U (0.6 ± 0.6), which is consistent with blooming organic productivity that likely led to more consumption of oxygen in the water column.