Trabeculae in vertebral bodies are unequally distributed within the cervical spine (CS), the thoracic spine (TS), and lumbar spine (LS). Such structures are also unequally distributed within the individual vertebrae. Exact knowledge of the microstructure of these entities could impact our understanding and treatment of fractures caused by osteoporosis and possibly improve surgical approaches. Appropriate investigations could help clarify the pathomechanisms of different forms of osteoporotic vertebral fractures, as well as different changes in morphological findings like the trabecular bone score (TBS). In the present study, we applied punctures to the craniocaudal and ventrocaudal directions and obtained cylinders of cancellous bone from the central portions and marginal regions of cervical vertebrae 5 and 6, thoracic vertebrae 8 and 12, and lumbar vertebrae 1 and 3. We systematically analyzed these samples to determine the bone volume fraction, trabecular thickness, separation, connectivity density, degree of anisotropy, and structure model index. Using an 8-gauge Jamshidi needle, we obtained samples from three quadrants (Q I: right margin; Q II: central; Q III: left margin) in the frontal and transverse plane and prepared these samples with a moist cloth in a 1.5 mL Eppendorf reaction vessel. The investigations were performed on a micro-CT device (SKYSCAN 1172, RJL Micro & Analytic Company, Karlsdorf-Neuthard, Germany). All collected data were analyzed using the statistical software package SPSS (version 24.0, IBM Corp., Armonk, NY, USA). Student's t test, the Wilcoxon-Mann-Whitney test, the Chi-squared test, and univariate analysis were used for between-group comparisons. The selection of the test depended on the number of investigated groups and the result of the Shapiro-Wilk test of normal distribution. In the case of statistically significant results, a post hoc LSD test was performed. In total, we obtained 360 bone samples from 20 body donors. The craniocaudal puncture yielded data of similar magnitudes for all investigated parameters in all three quadrants, with the highest values observed in the CS. Comparisons of the ventrodorsal and craniocaudal microstructure revealed a significantly lower trabecular density and a significantly higher degree of anisotropy in the craniocaudal direction. The results presented different distributions and behaviors of trabecular density, with lower density in the mid-vertebral region over the entire breadth of the vertebrae. Reduced trabecular density caused a higher degree of anisotropy and was, therefore, associated with a lower capacity to sustain biomechanical loads. Fractures in fish vertebrae were easily explained by this phenomenon. The different changes in these structures could be responsible, in part, for the changes in the TBS determined using dual-energy X-ray absorptiometry. These results confirm the clinical relevance of the TBS.