Pollen data have been recorded at Novi Sad in Serbia since 2000. The adopted method of producing pollen counts has been the use of five longitudinal transects that examine 19.64% of total sample surface. However, counting five transects is time consuming and so the main objective of this study is to investigate whether reducing the number to three or even two transects would have a significant effect on daily average and bi-hourly pollen concentrations, as well as the main characteristics of the pollen season and long-term trends. This study has shown that there is a loss of accuracy in daily average and bi-hourly pollen concentrations (an increase in % ERROR) as the sub-sampling area is reduced from five to three or two longitudinal transects. However, this loss of accuracy does not impact on the main characteristics of the season or long-term trends. As a result, this study can be used to justify changing the sub-sampling method used at Novi Sad from five to three longitudinal transects. The use of two longitudinal transects has been ruled out because, although quicker, the counts produced: (a) had the greatest amount of % ERROR, (b) altered the amount of influence of the independent variable on the dependent variable (the slope in regression analysis) and (c) the total sampled surface (7.86%) was less than the minimum requirement recommended by the European Aerobiology Society working group on Quality Control (at least 10% of total slide area).