Although exogenous application of glycinebetaine (GB) is widely reported to regulate a myriad of physio-biochemical attributes in plants under stressful environments including drought stress, there is little information available in the literature on how and up to what extent GB can induce changes in anatomical features in water starved plants. Thus, the present research work was conducted to assess the GB-induced changes in growth, physio-biochemical, and anatomical characteristics in two cultivars (CK-1 and F-411) of oat (Avena sativa L.) under limited water supply. After exposure to water stress, a considerable reduction was observed in plant growth in terms of lengths and weights of shoot and roots, leaf mesophyll thickness, leaf midrib thickness, root cortex thickness, root diameter, stem diameter, stem phloem area, and stem vascular bundle area in both oat cultivars. However, water stress resulted in a significant increase in leaf total phenolics, hydrogen peroxide (H2O2), ascorbic acid (AsA), GB contents, activities of enzymes (CAT, SOD and POD), total soluble proteins, leaf epidermis (abaxial and adaxial) thickness, bulliform cell area, sclerenchyma thickness, root endodermis and epidermis thickness, root metaxylem area, stem metaxylem area and stem sclerenchyma thickness in both oat cultivars. Foliar-applied 100 mM GB suppressed H2O2 contents, while improved growth attributes, free proline and GB contents, activity of SOD enzyme, leaf abaxial epidermis thickness, leaf bulliform cell area, leaf midrib thickness, leaf sclerenchyma thickness, root cortex thickness, root endodermis, epidermis thickness, root stele diameter, stem diameter, stem epidermis thickness, stem metaxylem area, and stem phloem and vascular bundle area in both oat cultivars. For both oat cultivars, CK-1 was superior to F-411 in leaf abaxial epidermis thickness, leaf mesophyll, leaf sclerenchyma, root metaxylem area, stem diameter, stem epidermis, sclerenchyma thickness, stem metaxylem area, and stem vascular bundle area. Overall, both oat cultivars showed inconsistent behavior to water stress and foliar-applied GB in terms of different physio-biochemical attributes, however, CK-1 was superior to F-411 in a number of anatomical features of leaf, root, and stem.