The scanning force microscope has been used to study the lattice and defect structures of multilayers of the unsaturated fatty acid, 12‐8 diacetylene (10,12‐pentacosadiynoic acid) in ambient conditions. Films were prepared by the Langmuir–Blodgett technique on ordinary microscope glass and indium tin oxide coated glass. Lattice structures were deduced from the well resolved molecular images and, before polymerization, were found to be nearly centered rectangular with lattice parameters (0.88±0.06) nm and (0.51±0.04) nm. After exposing to ultraviolet radiation for polymerization the lattice structure changed to an oblique lattice with lattice parameters (0.466±0.008) nm and (0.55±0.01) nm. Molecular level defects such as dislocations and grain boundaries were resolved in these films very clearly. Observation of these kinds of defects implies that it is possible to reliably image the real surface molecules under ambient conditions. Polymerization was found to take place in one of the lattice directions and the modulation perpendicular to that direction was more pronounced than along the polymer backbone.