Molecular markers have provided evidence that the canopy position from which open-pollinated seed is harvested in Eucalyptus globulus can markedly affect its outcrossing rate. Outcrossing rates were higher and more uniform in upper canopy samples. As expected, no significant differences in outcrossing rate were found between top and bottom canopy samples of self-incompatible trees, but differences were large between top and bottom samples of self-compatible trees. In one tree, 73% of the open-pollinated progenies at the bottom of the canopy were selfed compared to 26% at the top. Such selfing can severely reduce survival and growth. This result clearly indicates the importance of sampling position within the canopy when collecting open-pollinated seed for deployment or genetic evaluation.