This paper presents the results of a comparative analysis of relative tax burden of taxpayers (natural persons) with income from employment in the Czech Republic from 1993 to 2009. Single, childless taxpayers with tax residency in the Czech Republic were chosen to represent this category of taxpayers. The amount of the tax burden was conceived as the sum of natural person income tax and the social and health insurance premiums paid by the taxpayer (employee), constituting in its nature and character a tax equivalent. The amount of these legally imposed payments was set with respect to the state valid and effective as of December 31st of the relevant year, in the case of the year 2009 with respect to the state as of June 30th. The results of the performed analysis prove gradual decrease in relative tax burden in the period of 1993–2009 for taxpayers of all income groups. However, the decrease cannot be characterized as uniform. The most marked decrease was identified with taxpayers in the upper income groups. The most significant change in the relative tax burden took effect in association with changes introduced in the Act on Income Taxes as of January 1st, 2008 when the progressive income tax of natural persons was replaced with a flat-rate tax and when changes were introduced to the concept of the partial income tax base calculations applicable to natural persons. The main contribution of this paper can be seen in the fact that the comparison encompasses the time interval covering the entire effective life of the Act on Income Taxes. It is also important that in view of the specified solution conditions, the relative tax burden values presented in the graphs can be understood as maximum values of the tax burden of taxpayers having income from employment.