Implemented in road transport enterprises (RTEs) on a large scale, telematics systems are dedicated both to the particular aspects of their operation and to the integrated fields of the total operational functioning of such entities. Hence, a research problem can be defined as the identification of their efficiency levels in the context of operational activities undertaken by RTEs (including more holistic effects, e.g., lowering fuel/energy consumption and negative environmental impacts). Current research studies refer to the efficiency of some particular modules, but there have not been any publications focused on describing the efficiency of telematics systems in a more integrated (holistic) way, due to the lack of a universal tool that could be applied to provide this type of measurement. In this paper, an attempt at filling the identified cognitive gap is presented through empirical research analysing the original matrix developed by the authors that refers to the efficiency rates of organisational activities undertaken by RTEs. The purpose of this paper is to present a tool that has been designed to provide a holistic evaluation of efficiency of telematics systems in RTE operational management. The results are presented in a form of an individual (ontogenetic) matrix of the analysed companies, for which a determinant was calculated with the use of Sarrus’ rule. Obtained in such a way, the set of values identified for the determinants of the subsequent ontogenetic matrices came as an arithmetic progression that characterised the scope and the level of the influence exerted by the implemented IT (information technology) systems on the organisational efficiency of operational activities undertaken by the analysed RTEs. We present a hypothesis stating that the originally developed matrix can be viewed as a reliable tool used for comparative analysis in the field of efficiency of telematics systems in RTEs, and this hypothesis was positively verified during the research. The obtained results prove the significant potential for the wide application of the discussed matrix, which can be used as a universal tool for the analysis and comparison of efficiency indicated by the integrated IT systems in the operational activities undertaken by RTEs.