The self-assembly of a mixture of hexadecanoic acid with racemic (R)/(S)-2-bromohexadecanoic acid has been investigated using scanning tunneling microscopy. When this mixture is physisorbed onto the basal plane of graphite, hexadecanoic acid controls the resultant two-dimensional structure. By self-assembling on a graphite substrate into two distinct domains exhibiting nonsuperimposable mirror image morphology, achiral hexadecanoic acid induces the segregation of 2-bromohexadecanoic acid into enantiomerically pure (R and S) domains. The 2-bromohexadecanoic acid molecules provide convenient markers to directly differentiate the 2D chiral domains of inherently achiral hexadecanoic acid. The pattern in which hexadecanoic acid assembles with enantiomerically pure 2-bromohexadecanoic acid is shown to be more energetically favorable than a monolayer in which the unsubstituted fatty acid is mixed with both chiral isomers of 2-bromohexadecanoic acid.