Constructing a robust understanding of homologous chromosomes, sex chromosomes, and the particulate nature of genes is a notoriously difficult task for undergraduate biology students. In this lesson, students expand their knowledge of human chromosome pairs by closely examining autosomes, sex chromosomes, and the non-homologous elements of the human X and Y sex chromosomes. In this four- part guided activity, students will learn about the structure and function of human autosomal and sex chromosomes, view and interpret gene maps, and gain familiarity with basic bioinformatics resources and data through use of the National Center for Biotechnology Information (NCBI) website. (Student access to computers with Internet connectivity is required for the completion of all Investigations within this lesson.) By viewing chromosomes and gene maps, students will be able to contrast expectations for homologous autosomal chromosome pairs and sex chromosome pairs, as well as gain a deeper understanding of the genetic basis for human chromosomal sex determination. In the last part of this lesson, students can also begin to understand how genetic mutations can lead to sex-reversal. The lesson, as presented, is intended for an introductory biology course for majors, but could be modified for other audiences. In addition, each exercise (“Investigation”) within the lesson can be used independently of the others if an instructor wishes to focus on only a subset of the learning objectives and provide the necessary context.  Options to extend the lesson related to interpreting phylogenies, and contrasting definitions of sex and gender are also provided.