The literature has shown that sexual health literacy has limited applicability in many developing countries. The present study, therefore, aimed to develop and examine the validity and reliability of the Sexual Health Literacy Scale (SHLS) among a sample of 595 Iranian university students. The first analysis yielded themes obtained from a qualitative content analysis of the 118-item SHLS scale. Concepts were extracted using the method of latent content analysis (Bengtsson, NursingPlus Open 2:8-16, 2016). 327 initial codes were extracted and main categories (Elo and Kyngäs, J Adv Nurs. 62 (1): 107-115, 2007) or themes (Graneheim and Lundman, Nurse Education Today 24: 105-112, 2004) obtained consisting of the information source, individual barriers, understanding and application, capacity and motivation, damage, skills, sexual rights, and socio-cultural barriers. In the second analysis, the 595 students were randomly split into two groups. An exploratory factor analysis was conducted on the themes derived and quantified in Phase 1. 6 Factors were obtained and found to be consistent in both groups. Criterion-related validity of sexual health literacy was determined by stepwise multiple regression to predict marital satisfaction. The reliability of SHLS was also investigated. The third analysis examined the fit of the 6 factors obtained from the 595 students in the original sample to a new sample of 221 university students using cross-validation via confirmatory factor analysis. We developed and validated a six-factor structure of the Sexual Health Literacy Scale 106 (SHLS-106): factor 1, Sexual Skills; factor 2, Individual Socio-cultural Barriers; factor 3, Sexual Vulnerability; factor 4, Resources to Access Sexual Information; factor 5, Understanding and Application; factor 6, Capacity and Motivation. SHLS-106 shows good test-retest reliability and criterion, incremental and convergent validities. This is the first study to examine the validity and reliability of the Sexual Health Literacy Scale in an Iranian sample. Considering the acceptable validity and reliability of this instrument, the psychometric properties of SHLS-106 need to be further investigated in diverse, more extended samples to clarify the extent of application of this scale in different settings. SHLS-106 can effectively examine sexual health literacy, a dynamic scale in nature influenced by the individual, healthcare system, contextual, and social factors in different cultures.