Employee performance and their new ideas, as well as their efforts to promote the company in positive ways help build the values of an enterprise. Properly motivated managers, white-collar, and blue-collar workers use their performance to affect the business efficiency, and therefore the success and sustainability of the enterprise. Selecting the right structure of motivation factors, especially those aimed at job category and gender, is the main role of enterprise management. The aim of this study is to analyze and define differences in the perception of the preferred level of motivation in terms of gender and job category. The questionnaires were given to randomly selected employees working in Slovak enterprises in order to ensure variability and randomness of respondent selection which is necessary for relevant data acquisition. Following the average, the order of the importance of motivation factors of 3720 respondents was defined. The Student’s t-test and Tukey’s HSD test were used. We confirmed that there are statistically significant differences in the perception of the motivation in terms of job category. Moreover, we proved the significant differences between genders in the job category of blue-collar workers. We did not observe differences between genders in the other job categories studied. The results reported should be accepted and implemented in motivational programs by the employees of human resource departments as a way to keep up with strategic human resource management.