Pichia kluyveri is a maltose-negative yeast applied for non- and low-alcoholic beer (NABLAB) production due to its fruity aroma attributes, however, related literature is still limited. Thus, this study aims to investigate basic fermentation parameters, such as growth, sugar consumption, and production of primary fermentation metabolites, but also the amino acid preference of three different P. kluyveri strains in an agitated model brewers’ wort substrate. Furthermore, the effect of agitation on these parameters was studied for one of the strains. The three strains assessed, PKCH_01, PKCH_02 and PKCH_03, shared a glucophilic nature, a cell growth from 4E+07 to 6E+07 E+07 CFU/mL, similar amino acid preference, and equivalent ethanol yield factor (Y_se) from 0.45 to 0.5 g ethanol/g glucose. In addition, agitation increased the cell growth yield factor by 78% and favored the uptake of amino acids, minimizing their release to the medium. Interestingly, a strong preference for methionine (above 80% consumption) by P. kluyveri in a beer-related medium was reported for the first time, whereas glutamine and glutamate were within the low-preference group (below 15% consumption). This study provides a better understanding about fermentation and physiological attributes of P. kluyveri, thereby revealing its potential for brewing applications.