In Australia, periodic waterlogging throughout the cotton growing season can cause production losses of up to 10%. There is limited information on the genetic variation in cotton for waterlogging tolerance. The aim of this study was to identify methods to evaluate physiological responses under waterlogging conditions that may lead to identifying waterlogging tolerant and sensitive cotton cultivars. A field experiment was conducted in Narrabri, north-western New South Wales using thirteen upland cotton (Gossypium hirsutum L.) cultivars (Georgia King, McNair 1032, PD93057, LA 887, Codetec 401, DP 16, DP 90, Coker 315, CIM 443, Gohar 87, Sicot 71, Sicot 73 and Sicot 80) and one Gossypium barbadense cultivar (Pima A-8) originating from diverse environmental regions. Parameters measured to assess response to waterlogging included: SPAD (leaf colour) readings, leaf nutritional status, leaf photosynthetic rate, plant and root morphology, and final yield. Leaf SPAD readings, nitrogen and potassium concentrations were reduced in waterlogged treatments compared to the respective controls, and varied with cultivar. Leaf phosphorus, calcium, magnesium, manganese and sulphur concentrations were reduced in the waterlogged treatment compared to the respective controls in all cultivars. Waterlogging increased leaf total iron concentration in all cultivars. No aerenchyma on cotton roots were observed in this study. Leaf SPAD readings, nitrogen and potassium concentrations suggested that the most waterlogging tolerant cultivars were Gohar 87, Pima A-8, Sicot 71, Sicot 73 and Sicot 80 which originate from production on heavy clays, and the most susceptible were Georgia King, LA 887, DP 16, DP 90 and CIM 443 which originate from production on lighter texture soils. This study helped to target those measurements that may be of most use to screen for waterlogging tolerance.