The study investigated patterns of students’ ability derived from achievement data in Biology classroom characterized by assertive questioning strategy in Benue State, Nigeria. The study was undertaken as a result of persistent poor performance of students in external examinations, and the need to find out if assertive questioning instructional strategy will improve the performance of students of varied abilities in the study area. Five research questions were raised and answered as guide to the study, while five hypotheses were formulated and tested. Quasi-experimental research design was adopted. The population comprised 5300 senior secondary two (SSII) students, from which a sample of 345 students were selected from six public schools using multistage sampling technique. Data was generated using Ability Group Test (AGT) and Biology Achievement Test (BAT). The various instruments were validated and subjected to reliability analysis using Kuder-Richardson formula 21 (KR21) to determine their reliability coefficients. Data collected were analyzed using means, bar graphs and standard deviations to answer the research questions while Analysis of Covariance (ANCOVA) was used to test the hypotheses at 0.05 level of significance. The results showed among others that there is significant difference in the mean achievement scores of students taught cellular respiration using assertive questioning instructional strategy and lecture method ( F (1,342) = 49.330; p = 0.000 < 0.05); there is significant difference in the mean achievement scores of male and female students taught cellular respiration using assertive questioning instructional strategy (F(1,194) = 28.871; p = 0.000 < 0.05); and that there is significant difference in the mean achievement scores of students of varied abilities taught cellular respiration using assertive questioning instructional strategy (F (1,192) = 23.707; p = 0.000 < 0.05). Based on the findings of the study, it was recommended that Biology teacher-trainees should be trained on the use of the assertive questioning strategy; Seminars and workshops should be organized for Biology teachers in secondary schools to enable them to use assertive questioning strategy in the classroom and Biology teachers should endeavour to give male and female students equal learning opportunities in the classroom. Teachers are advised to adopt assertive questioning strategy as the method has the capacity to enhance male and female students of varied abilities’ achievement in Biology.