Published reports of comparison of erythrocyte parameters for newly enrolled urban and rural junior secondary school students in different geographical zone in Nigeria are relatively scanty. To determine and compare erythrocyte parameters of newly enrolled urban and rural junior secondary school students in south-south and south-east, Nigeria. The study population was a total of 240 subjects (60 male and 60 female subjects for each urban and rural area) of JSS1 students. 5mls of blood sample were collected from ante cubital vein with minimum stasis and the values of Red blood cell count, Haemoglobin, Mean cell volume, Mean cell haemoglobin, Mean cell haemoglobin concentration, Red cell distribution coefficient volume, Red cell distribution standard deviation and Haematocrit, was determined by MAXCOM MC- 6200 fully automated haematology analyzer, while the data were analyzed using Z-test; p value less than 0.05 were considered statistically significant. There were significant gender variations in the values of these parameters in both urban and rural newly enrolled students. The mean values of Haemoglobin, Mean cell volume, Mean cell haemoglobin, Red cell distribution width standard deviation and Haematocrit were significantly higher in rural males and females compared to urban males and females (p˂0.05). No significant differences were found in mean values of red blood cell count and Coefficient variation of red cell distribution width when compared both rural and urban males and females. From the above study, there is variation in the gender, age and some erythrocyte parameters amongst newly enrolled urban and rural junior secondary school students under investigation.