Purpose: Atherosclerosis is a multi-factorial disease occurring in vascular sites subjected to complex flow; such as arterial bifurcations experiencing Oscillatory shear stress (OSS) and curvatures where Low shear stress (LSS) occurs. The behaviour and phenotype of macrophages, the most abundant innate immune cell in atherosclerosis, is crucial in determining the outcome of atherosclerotic plaque formation. Macrophage behaviour is conditioned by their polarisation towards a pro-inflammatory phenotype (called M1) or a regulatory one (broadly named M2). The aim of this study is to establish the activation and polarisation status of macrophages in different plaque phenotypes and shear stress conditions. Methods: We utilised a murine model which imposes LSS to develop vulnerable plaques, and OSS to induce a stable plaque phenotype. A perivascular shear stress modifying cast was tied around a carotid artery mimicking LSS and OSS in ApoE-/- mice fed a high fat diet (HFD) from 17 weeks of age. We examined the cellular protein expression of the pan-macrophage marker CD68 and polarisation markers iNOS (M1), CD206 (M2) and HO-1 (Mox subset) in the aortic root and cast-treated carotid artery by Immunohistochemistry and Confocal Immunolocalisation at early (12 weeks), intermediate (28 weeks), and late stages of atherosclerosis (53 weeks). Results: In the aortic roots, the majority of CD68+ cells stained positively for HO-1, reaching a maximum at 28 weeks (36.3±1.2% of lesion area; p<0.001 vs. 12 and 53 weeks). Immunopositivity for iNOS and CD206 were confined to a small proportion of macrophages in aortic roots (max 5.6±2.3% and 4.2±1.1% of lesional area at 20 weeks respectively). In stark contrast, the majority of macrophages in the LSS-induced carotid artery lesions express iNOS (23.6±5.1% vs. 5.4±2.7% lesion area; p=0.006). The expression of CD206 and HO-1 instead was not different between LSS and OSS. Conclusions: The majority of macrophages in early and intermediate lesions of the aortic root express HO-1 (Mox phenotype). M1 or M2 polarised macrophages are present in smaller numbers at early and intermediate stages of disease but not in late disease. In atherosclerotic lesions of the carotid artery, M1 macrophages are significantly more prevalent in the low shear stress-modulated plaque. These differences suggest low shear stress promotes polarisation towards the M1 macrophage phenotype.