The Bachelor thesis deals with the issue of human resources, especially employee motivation in the company specialised in rail transport. Following the data gathered in the year 2003, 2009, 2016, 2019 and 2020, the level of worker motivation is defined. Data were gathered using a questionnaire with close-ended questions. Data evaluation provides the information necessary to define the most important motivation factors in individual analysed years. Subsequently, statistically significant differences at the level of significance α = 5 % were defined using the ANOVA (Duncan test. The findings that statistical significant differences in the level of worker motivation emerged in the company specialised in rail transport can be considered the result of the Bachelor thesis. Mentioned statement can be stated with the 99 % probability. Following our research the fact that significant changes in the structure of motivation factors as well as the change in the importance of motivation factors occurred during the analysed period can be seen. Mentioned changes are associated with the eight most important motivation factors (career advancement, atmosphere in the workplace, good team at work, selfactualisation, workplace security, fringe benefits, communication in the workplace, supervisor’s approach) determined according to the order of importance in the year 2020. Further two motivation factors (name of the company, recognition) were determined following the professional knowledge. During 12 months, we suppose to analyse motivation factors again in order to find further changes in the structure of motivation factors after incorporating steps to improve motivational climate in the company. In the case of further significant changes, motivation programme for employees reflecting the requirement of employees must be created.