A scanning second harmonic generation (SHG) microscopy technique in conjunction with a polarization resolved detection scheme is applied to probe the local molecular organization in organic monolayers. A full SHG polarization analysis is developed to investigate local effects such as orientational heterogeneities and random orientations in molecular aggregates. The SHG signal measured in hemicyanine-based monolayers deposited on glass substrates evidences spatial micrometric-size domains where the non-centrosymmetric molecular alignment is modeled with a high accuracy based on polarization information. In addition, an incoherent and spatially uniform SHG emission is measured in monolayers displaying molecular aggregation. (c) 2005 Elsevier B.V. All rights reserved.