Cell wall composition, pectin, and hemicellulose fine structure variation were assessed in peach and related genotypes with contrasted texture and fruit shape. Cell walls were prepared from four commercial peaches, eight genotypes from the Jalousia x Fantasia peach cross, and six genotypes from the Earlygold peach x Texas almond cross. Sugar composition was determined chemically while fine structure of homogalacturonan pectin and xyloglucan hemicellulose were assessed by coupling pectin lyase and glucanase degradation, respectively, with MALDI-TOF MS analysis of the degradation products. The results indicate clear compositional and structural differences between the parents and their related genotypes on the basis of pectin versus cellulose/hemicellulose content and on the fine structure of homogalacturonan and xyloglucan. A relation between methyl- and acetyl-esterification of pectin with fruit shape is revealed in the Fantasia X Jalousia peach genotypes.