Motivation is an inner force that activates and provides direction to our thought, feelings and actions. Two main characteristics of motivation are goal directed behavior and persistence. Motivated people persistently work for the goal till it is achieved. Intrinsic-motivation, self-efficacy and assessment anxiety, independence, career motivation and grade motivation are motivational elements that become visible to affect one’s regulated learning, presented by Glynn and Koballa (2006) Science motivation questionnaire (SMQ) administered its construct validation was investigated on students at the college level. The authors recommended that it may be tried out on students with science major in other cultured settings to examine its construct validity. The study, therefore, sought to explore its construct validity for science students at the college level along with their gender wise comparison on science motivation. The research questions were what evidence is there that motivation can be measured through the SMQ validity and reliability? Are there gender differences in motivation with science students? Are there differences in motivation with science learning experience? The objectives of the study were thus to investigate the construct validity of the science motivation questionnaire, to examine any motivational differences between male and female intermediate science students, to examine any differences between first and second year science students, and to put forward recommendations in the light of the findings of the study. The college students of intermediate classes in the province of Punjab constituted the population of the study. A sample of 600 1st and 2nd year science students served as the sample of the study which were selected through two stage cluster sampling. To measure the science motivation components, the 30-item Likert-type science motivation questionnaire developed by Glynn and Koballa (2006) was used in the study that claims to influence self-regulated learning. Principal Components Analysis, using varimax rotation, was used to find the fundamental structure of the SMQ. In addition, inter-item correlation was explored using Kendall’s Tau-b. Chi-Square ‘contingency test’ was applied to compare the two distributions of responses. EXCEL spread sheet was used for chi-square analysis because SPSS does not handle it well. The Scree plot did not indicate any factor structure; also inter-item correlations (Kendall’s Tau-b) suggested no factor structure. However, in gender comparison, the 30 items, there are statistical differences in 9 of them.