There has always been a specific focus on nitrogen fertilization in sugar beet production due to its important effect on sugar beet root yield and quality. For stable sugar beet growth and satisfactory root yield and quality, balanced N fertilization is crucial. Thus, this study aimed to investigate spring N fertilization in two seasons as the following treatments: N0—control, N1—only pre-sowing fertilization, and N2—pre-sowing with topdressing. Four different genotypes were included in the study (Serenada, Colonia, Fred, and Danton). The experiment was set up in a plain area, belonging to the temperate climate zone in Eastern Croatia (Županja and Vrbanja), with the long-term mean (LTM) (March–October) air temperature around 16 °C and the total precipitation of 515 mm. Pre-sowing N fertilization had a smaller impact on root yield in the year with higher precipitation (31% higher than LTM). Therefore, the average yields with pre-sowing fertilization (N1) and pre-sowing fertilization with top dressing (N2) were very similar and were only 7% higher than those of the control. In a season with less rainfall (29% less than LTM), pre-sowing fertilization with top dressing (N2) had a more pronounced effect on the increase in sugar beet root yield, which was 17% higher compared to that of the control treatment. The sugar beet sucrose content and quality parameters (brei impurities, loss of sugar in molasses, extractable sugar) differed when N fertilization was applied among locations in both seasons. The white sugar yield was the highest at N2 treatment with pre-sowing and topdressing N fertilization. In general, according to the average of all locations and years of research, the Serenada hybrid achieved the highest average root yield (81.1 t ha−1), while Colonia exhibited the highest root sugar content (14.5%) and white sugar yield (9.7 t ha−1).