The experimental approach and mechanism of pressure tuning (PT) are introduced for the first stage of a comprehensive two-dimensional gas chromatography (GC × GC) separation. The PT-GC × GC system incorporates a first dimension (1D) coupled column ensemble comprising a pair of 1D columns (1D1 and 1D2) connected via a microfluidic splitter device, allowing variable decompression of carrier gas across each 1D column, and a conventional 2D narrow bore column. By variation of junction pressure between the 1D1 and 1D2 columns, tunable total 1D retentions of analytes are readily derived. Separations of a standard mixture comprising a number of different chemical classes (including alkanes, monoaromatics, alcohols, aldehydes, ketones, and esters) and Australian tea tree oil (TTO) were studied as practical examples of the PT-GC × GC system application. This illustrated the change of analyte retention time with experimental conditions depending on void time and retention on the different columns. In addition to void time change, variation of carrier gas relative decompression in the 1D ensemble leads to tunable contribution of the 1D1/1D2 columns that changes apparent polarity and selectivity of the ensemble. The resulting changes in 1D elution order further altered elution temperature and thus retention of each analyte on the 2D column in temperature-programmed GC × GC. 2D orthogonality measurements were then conducted to evaluate overall separation performance under application of different 1D junction pressure. As a result, distribution and selectivity of particular target compounds, monoterpenes, sesquiterpenes, and oxygenated terpenes in 2D space, and thus orthogonality, could be adequately tuned. This indicates the potential of PT-GC × GC to be applicable for practical sample separation and provides a general approach to tune selectivity of target compounds.