The relative fluorescence of acritarchs (400–700 nm range) was investigated as an alternative technique of determining the level of thermal maturity of marine Paleozoic source rocks in Canada. The sedimentary strata examined include the Blue Mountain, Lindsay (Collingwood Mbr), Guelph (Eramosa Mbr) and Marcellus formations from southern Ontario, Yeoman and Winnipegosis formations from Saskatchewan, as well as the Manitoba and Elk Point groups from Saskatchewan and Alberta, respectively. The examined strata contain oil-prone, predominantly marine organic matter (Type II and I kerogen) with varying proportions of bituminite and alginite as the dominant maceral components. Acritarchs, which occur as persistent maceral inclusions within such organic facies, show excellent potential in thermal maturity estimations. The fluorescence of acritarchs shows a progressive red shift throughout the initial and main stages of oil generation with a trend parallel to that of alginite. However, as a result of their lower sensitivity to increasing burial temperature, the λmax and Q values of acritarchs are lower than the corresponding λmax and Q of Gloeocapsomorpha prisca and Leiosphaeridia alginite, with the difference becoming more pronounced with increasing thermal maturity. Correlations with optical and geochemical maturity indicators, such as reflectance of chitinozoa (%ChR o), bitumen (%BR o) and vitrinite (%VR o), Rock-Eval Tmax and the isomerization ratio of regular steranes (S/[S+R], ββ/[αα+ββ]), indicate that in kerogen Type II organic matrix both λmax and Q values of acritarchs vary little until the onset of oil generation. At this maturity level, corresponding to VR o<0.5% and Tmax<435°C, λmax values are commonly 450 nm or less whereas Q is below 0.5. Within the zone of the initial phase of oil generation the λmax shows a steady increase to 480 nm, and then a more rapid raise throughout the oil window (520–550 nm). These changes in λmax are not accompanied by a meaningful increase in Q values which, in general, remain around 0.5. In kerogen Type I organic matrix no significant variations in λmax and Q have been observed up to a maturity level corresponding to VR o⩽1% and Tmax up to 460°C.